Juvenile Methoxychlor Exposure on Adult Rat Nervous, Immune, and Reproductive System Function

FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.

40, 138–157 (1997)

FA972381

The Effects of Perinatal/Juvenile Methoxychlor Exposure on Adult Rat Nervous, Immune, and Reproductive System Function R. E. Chapin,* M. W. Harris,* B. J. Davis,† S. M. Ward,† R. E. Wilson,† M. A. Mauney,‡ A. C. Lockhart,‡ R. J. Smialowicz,§ V. C. Moser,Ø L. T. Burka,\ and B. J. Collins\ With the Technical Assistance of E. A. Haskins, J. D. Allen, L. Judd, W. A. Purdie, H. L. Harris, C. A. Lee, and G. M. Corniffe *Reproductive Toxicology Group, National Toxicology Program, †Laboratory of Experimental Pathology, and \Chemistry Group, NIEHS, MD B3-05, P.O. Box 12233, RTP, North Carolina 27709; ‡Analytical Sciences, Inc., 2605 Meridian Parkway, Suite 200, Durham, North Carolina 27713; and §Experimental Toxicology Division and ØNeurotoxicology Division, National Health and Environmental Effects Laboratory, U.S. EPA, RTP, North Carolina 27711 Received July 10, 1997; accepted September 11, 1997

The Effects of Perinatal/Juvenile Methoxychlor Exposure on Adult Rat Nervous, Immune, and Reproductive System Function. Chapin, R. E., Harris, M. W., Davis, B. J., Ward, S. M., Wilson, R. E., Mauney, M. A., Lockhart, A. C., Smialowicz, R. J., Moser, V. C., Burka, L. T., and Collins, B. J. (1997). Fundam. Appl. Toxicol. 40, 138–157. In order to address data gaps identified by the NAS report Pesticides in the Diets of Infants and Children, a study was performed using methoxychlor (MXC). Female rats were gavaged with MXC at 0, 5, 50, or 150 mg/kg/day for the week before and the week after birth, whereupon the pups were directly dosed with MXC from postnatal day (pnd) 7. Some dams were killed pnd7 and milk and plasma were assayed for MXC and metabolites. For one cohort of juveniles, treatment stopped at pnd21; a modified functional observational battery was used to assess neurobehavioral changes. Other cohorts of juveniles were dosed until pnd42 and evaluated for changes to the immune system and for reproductive toxicity. Dose-dependent amounts of MXC and metabolites were present in milk and plasma of dams and pups. The high dose of MXC reduced litter size by É17%. Ano-genital distance was unchanged, although vaginal opening was accelerated in all treated groups, and male prepuce separation was delayed at the middle and high doses by 8 and 34 days, respectively. In the neurobehavioral evaluation, high-dose males were more excitable, but other changes were inconsistent and insubstantial. A decrease in the antibody plaque-forming cell response was seen in males only. Adult estrous cyclicity was disrupted at 50 and 150MXC, doses which also showed reduced rates of pregnancy and delivery. Uterine weights (corrected for pregnancy) were reduced in all treated pregnant females. High-dose males impregnated fewer untreated females; epididymal sperm count and testis weight were

This article has been reviewed by the National Health and Environmental Effects Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. 0272-0590/97 $25.00 Copyright q 1997 by the Society of Toxicology. All rights of reproduction in any form reserved.

AID

FAAT 2381

/

6k23$$$281

reduced at the high, or top two, doses, respectively. All groups of treated females showed uterine dysplasias and less mammary alveolar development; estrous levels of follicle stimulating hormone were lower in all treated groups, and estrus progesterone levels were lower at 50 and 150 MXC, attributed to fewer corpora lutea secondary to ovulation defects. These data collectively show that the primary adult effects of early exposure to MXC are reproductive, show that 5 mg/kg/day is not a NO(A)EL in rats with this exposure paradigm (based on changes in day of vaginal opening, pubertal ovary weights, adult uterine and seminal vesicle weights, and female hormone data) and imply that the sites of action are both central and peripheral. q 1997 Society of Toxicology.

In 1988, Congress asked the National Academy of Sciences to evaluate the likely risk posed to infants and children by residual pesticides in the food supply. This evaluation was published in 1993 (NAS, 1993). Among the recommendations were changes in the risk assessment process, changes in surveillance of the food supply, and a call for better information on the effects of pesticides on the developing animals’ reproductive, immune, and central nervous systems. The National Toxicology Program, in conjunction with collaborators at EPA’s National Health and Environmental Effects Research Laboratory, developed a design that would address some of the data needs identified in the NAS report. The NAS report identified the exposure period of concern for humans as being from the last trimester of pregnancy to 18 years of age, a range that is approximated in the rodent study used here. Because the concern was on direct consumption of pesticide residues, we dosed dams for the week before and the week after birth, and then direct-dosed the pups from postnatal day (pnd)7 to pnd42, the approximate age of puberty in these rats. Animals are taken at various points in the dosing period to ascertain effects: compound in milk is determined at pnd7, the last day of dosing for the dam, and the time during her dosing when she is likely to

138

11-10-97 09:54:57

ftoxal

139

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE

have the most milk available. A necropsy at the end of dosing (pnd 42) is intended to provide baseline data against which to compare any findings in the adults. That is, if there are juvenile effects, are these still there in the adults? How do any effects in the young affect adult function? The battery of neurotoxicity measures included in this design can identify treatment-related changes in sensory, motor, autonomic and cognitive function (Tilson and Moser, 1992). The functional assays employed for the immunotoxicity tests are sensitive at predicting immunotoxicants in rodents, using measures from the NTP’s Tier I group of assays and the most sensitive measures from Tier II (Luster et al., 1988, 1992). The reproductive endpoints focus on the development and function of the reproductive system and correlate necropsy gamete measures and histopathology with the functional evaluations. More information on the design and attendant rationale, as well as the chemicals intended to be tested and some additional background, has been published (Chapin et al., 1996; EHP, 1994). Some preliminary results of this study have been summarized and presented (Harris et al., 1996). This study is the first of several anticipated studies intended to generate a body of information on the susceptibility of young animals to the effects of a variety of different pesticides. Methoxychlor was the first compound to be examined in this design for three reasons: (1) it was expected to be a known positive for the reproductive endpoints, based on the large amount of previous data indicating adverse effects on reproductive function after early exposure (Tullner and Edgcomb, 1962; Harris et al., 1974; Gray et al., 1989), (2) it is one of the remaining 4 chlorinated pesticides still (at this writing) approved for use in the United States and (3) it addresses some priority data needs identified by Agency for Toxic Substances and Disease Registry (ATSDR, 1995). First synthesized more than 100 years ago (in Kapoor et al., 1970), methoxychlor (MXC) has been used for nearly 50 years for insect and larval control. Its advantages over DDT are that MXC is more readily metabolized and excreted by mammalian systems (Kapoor et al., 1970), and therefore there is less bioconcentration than with DDT. This metabolism also yields mono- and bis-hydroxy metabolites of MXC (Bulger et al., 1985) that are more estrogenic than the parent compound (Bulger et al., 1978), which helps explain both the uterotrophic effects noted earlier for MXC (Tullner, 1961) and the observation that MXC in vivo reduced the uterine uptake of radiolabeled estradiol (Welch et al., 1969). This biological activity has been thoroughly described in a large and exemplary corpus of work from Gray and colleagues (see, for example, Gray et al., 1989; Goldman et al., 1986; Cummings and Gray, 1987; Cummings, 1993), which stemmed from earlier observations (Hodge et al., 1950) that younger animals appeared more affected by MXC exposure than were older animals. This is at least in part due to the hormonal ‘‘imprinting’’ that occurs in the rodent

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

reproductive system during the last week of gestation and the first week postpartum (Chung and MacFadden, 1980). This leads to the concept that changes in hormonal profiles or activity during development will have lasting effects on target organs even as adults. Also, the estrogen receptor is present in numerous tissues in both the male and the female reproductive systems (rev. in Greco et al., 1993). Any MXC or active metabolite in the fetus or neonate, as inferred earlier (Appel and Eroschenko, 1992), would be expected to produce effects with the present design at lower doses than reported for studies that used older animals. METHODS Chemical Methoxychlor was obtained from Sigma Chemical Co. (St. Louis, MO) as a single batch. Neat MXC was identified by infrared and NMR spectroscopy and was found to be 95% pure; six minor peaks each accounted for 0.12 to 2.53% of the total area. MXC was dissolved in corn oil so that the desired dose was delivered at a dose volume of 5 ml/kg. MXC was weighed into a volumetric flask, corn oil added, and the contents stirred and sonicated for 30 min. Dosing solutions were analyzed for MXC content by gas chromatography using a capillary column and electron capture detection. All dosing solutions were stored at 47C and were found to be within 10% of target concentration. Animals and Conditions Rats were purchased as adults (70–90 days of age, females were received time-mated) from Taconic Farms (Tac:N(SD)fBR). All procedures were approved by the Institutional Animal Care and Use Committee and followed the PHS Guide stipulations (NIH, 1996). Animals were housed appropriately (1, 2, 3, or 4, depending on size and age) in polycarbonate cages with 12:12 hr light:dark cycles, 50{10% humidity, and an ambient temperature of 20{17C. Dams were distributed into groups by computer-generated stratified randomization to equalize body weight means across groups. All animals were allowed ad lib access to NIH-07 certified feed (Zeigler Bros., Inc., Gardners, PA) and deionized water. Naive adults destined for mating with adult MXC-treated partners were allowed an acclimation period of a 7–10 days prior to mating; the pregnant dams subject to prenatal dosing at the beginning of the study were acclimated for É7 days before dosing. Design, Groups, and Endpoints Logistical constraints in personnel and animal space required that two cohorts of dams were dosed: the first cohort produced pups for the milk analyses, the neurotoxicity and the immunotoxicity evaluations (Groups A, D, and G, Fig. 1). The second cohort of dams produced young for the other groups. Thus, the n for different endpoints varies slightly: newborn number, weight, and anogenital distance (AGD) data were collected on all pups; only the neurotox animals did not contribute data on vaginal opening and preputial separation. Only the reproductive group was evaluated for estrous cyclicity by vaginal lavage during maturation. Group I rats were subject to vaginal lavage as adults to allow correlation of the stage of the estrous cycle at necropsy with blood hormone levels. The design is schematized in Fig. 1. Time-mated, pregnant dams were dosed daily from gestation day 14 to pnd7, whereupon the litters were standardized to eight (four of each sex, when possible) and remained with the dam until weaning at pnd21. The pups were individually dosed from pnd7 until pnd42. A number of different groups were generated by this dosing scheme; they will be discussed chronologically and are listed on Fig. 1. At birth, the total number of pups and their individual weights were

ftoxal

140

CHAPIN ET AL.

FIG. 1. Schematic diagram of the design; see Methods for details.

recorded, as well as anogenital distance (Clemens et al., 1978) and the observations of any external malformations. Each pup was marked on pnd1 by toe tattoo; an individual identification number was made by tail tattoo at weaning on pnd21. Body weights were collected daily until the end of dosing. These animals comprised Groups B–I. Group A: Lactational Assessment Because the pups are not dosed until pnd7, there is the possibility of lactational transfer of MXC and/or metabolite. To develop a minimal data set on this, an additional eight dams (Group A) were treated for the 7 days before and after birth, as above. Early on pnd7, the pups were removed from the cage and the dam received the appropriate daily dose of vehicle or MXC. Three hours later, the dams were anesthetized with 50 mg/kg ketamine/ xylazine, following the general methods of Dostal et al. (1990). When anesthesia was achieved, the dams received 1 IU oxytocin in water i.p., and milk was manually expressed. Milk endpoints included quantity, proportion of total volume as lipids, and measures of total protein, triglycerides, and lactose content (as in Dostal et al., 1990). MXC and metabolites were analyzed as described below. Metabolites were analyzed due to the increased estrogenic activity of the hydroxyl metabolites (Bulger et al., 1978). Dams’ milk and plasma and pup plasma were analyzed for MXC and major metabolites: monohydroxy MXC, dihydroxy MXC, MXC olefin, monohydroxy MXC olefin, and dihydroxy MXC olefin. Plasma samples were diluted 1:1.25 with 5% methanol/water and extracted over a 3-ml. C18 column, washed with water followed by 40% methanol, and dried under

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

vacuum. Samples were eluted with 2 ml acetone, dried, reconstituted in acetone, and analyzed on a Hewlett Packard 5890 gas chromatograph with an electron capture detector. Mirex was used as the internal standard. Milk samples were processed similarly, except that matrix effects required each milk sample to be diluted 1:39 with pooled control rat plasma prior to analysis. The experimental limit of quantitation (ELOQ) was 5 ng/ml in plasma and 200 ng/ml in milk, due to the requirement for dilution. When levels in milk were below the ELOQ, the sample was diluted with less plasma, yielding an ELOQ of 50 ng/ml; not all samples had sufficient volume to allow such repetition. Group B animals were not evaluated for this study. Group C animals were eight nonlittermate males/dose level that were directly dosed from the end of dam dosing (pnd7) until asphyxiation on pnd21. Testes were removed and fixed in Bouin’s for possible histologic examination. These data were collected and are being reported in a separate manuscript. Group D: Neurotoxicity Assessments For the neurotoxicity segment, dosing stopped at pnd21, at which time most of the brain’s rapid growth has ceased (Benjamins and McKhann, 1981). The tests used to evaluate neurological function were those recommended for hazard identification by the U.S. EPA (1991). One male and one female pup from each litter was transferred to the NHEERL laboratories for subsequent behavioral testing. In the NHEERL facility, rats were singly housed in polycarbonate cages with hardwood chip bedding, in a room maintained

ftoxal

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE at 72{27F and 55{5% humidity. Food (Purina Rat Chow 5002) and deionized tap water were freely available except during testing. Treatment groups for neurotoxicity testing included vehicle (n Å 11/sex), 5 mg/kg (n Å 10/sex), 50 mg/kg (n Å 10/sex), and 150 mg/kg (n Å 6 males, n Å 7 females). Neurobehavioral integrity was evaluated using a series of observations and manipulations, termed a functional observational battery (FOB). Motor activity was also measured using an automated activity apparatus. Detailed descriptions of the procedures and scoring criteria have been published elsewhere (McDaniel and Moser, 1993; Moser et al., 1988). Rats were observed in the home cage, and atop a laboratory cart. Observations included evaluations of activity (e.g., rearing), reactivity, autonomic integrity (e.g., lacrimation), abnormal motor movements (e.g., tremors), neuromuscular function (e.g., gait characteristics), vestibular capability (e.g., righting reflex), and sensorimotor reactions (e.g., responses to various stimuli). Forelimb and hindlimb grip strength, landing foot splay, rectal temperature, and weight were also measured. Shortly after completion of these tests, the rat was placed in a photocell-based activity device in the shape of a figure-eight (Reiter, 1983) for 1-h sessions. The same observer conducted all portions of the study and was blind to the treatment conditions of each rat. FOB and motor activity testing took place over 2 days, with the treatment counterbalanced across squads of rats. Rats were tested a total of three times: pnd31–32 (half of the rats tested each day), pnd47–48, and pnd66–67. Passive avoidance was used as a general test of cognitive function. A shuttle box with an electrified grid toggle floor was connected to a precisionregulated shocker (Coulbourn Instruments, Allentown, PA). A guillotine door separated the two compartments, one side was lined on the outside with black posterboard (dark side), and the other side was illuminated with a high-intensity lamp. The rat was placed into the light side; after a 30-s delay, the door was opened. When the rat crossed all four feet into the dark side, the door shut and a 3-s, 1 mA shock was immediately delivered. Latency to cross into the dark side was recorded, and the rat was returned to the home cage. Specification of the light side was the same for each rat across testing, but alternated between rats. Retention at 24 h and 2 weeks was measured by again placing the rat into the box and measuring the latency to cross into the dark side. Retention tests were limited to 5 min. This training test and 24-h retest took place on pnd38–40. Avoidance retention was measured again 2 weeks later, on pnd54–55. Group E was killed by asphyxiation on pnd35 after maternal exposure as above, and direct pup exposure from pnd7 to pnd35. Tissues were prepared as for Group C and are undergoing evaluation and reporting with Group C tissues.

141

ployed were the T cell mitogens phytohemagglutinin (PHA-P; BurroughsWellcome, Research Triangle Park, NC) and concanavalin A (Con A; Difco Lab., Detroit, MI) and the B cell mitogen Salmonella typhimurium mitogen (STM; Ribi Immunochem, Hamilton, MT). The data are reported for the optimal concentration of each mitogen (i.e., 0.025 mg/culture of Con A, 1.0 PHA, and 20 mg/culture of STM). The one-way mixed lymphocyte reaction (MLR) was performed as described by Smialowicz et al. (1989) using responder (control or MXCdosed rats) and stimulator (pool of three male and three female untreated CD rats) lymph node lymphocytes. Stimulator cells (5 1 105), treated with mitomycin C (Sigma), were added to quadruplicate microtiter wells containing 2 1 105 responder cells. MLR cultures were incubated for 96 h at 377C and 5% CO2 . Twenty-four hours prior to harvest, cultures were pulsed with 0.5 mCi/well [3H]TdR. The results are expressed as the mean net CPM, subtracting the CPM of responder-only cultures from the CPM of responder plus stimulator cultures. Natural killer (NK) cell activity of splenocytes from rats dosed with MXC was determined in a 4-h 51Cr release assay (Smialowicz et al., 1987). 51 Cr-labeled YAC-1 mouse lymphoma cells, free of mycoplasma, were used as target cells in this assay. Percent specific 51Cr release was calculated using the formula [(E 0 S/(T 0 S)] 1 100, where E is the 51Cr released from target cells in the presence of spleen cells, S is the spontaneous release of 51Cr from target cells alone, and T is the maximum release of 51Cr from target cells in the presence of 0.25% Triton X-100. Spontaneous release was typically less than 10% of maximum releasable 51Cr activity.

This group was a subset of the remaining animals, killed shortly after the end of dosing, to ascertain if any effects were present at the end of treatment. In this study, these animals were killed on pnd46 and 47 and were of variable number/group. During maturation, measures were made of the day of first vaginal opening, and preputial separation (Korenbrot et al., 1977) on these early-necropsy animals and on the remaining male and female per litter (n Å 15/ dose level), which were used for reproductive assessments (Groups H and I, below).

Antibody plaque-forming cell (PFC) response and splenic phenotypes. Separate groups of MXC-exposed 9-week-old male and female rats (six/ group, except no 150 MXC males were available, due to reduced fertility at that level) were immunized with a single intravenous injection of 0.5 ml of 10% sheep red blood cells (SRBC, Environmental Diagnostics, Inc., Burlington, NC) in sterile saline. Four days later the rats were euthanized, weighed, and single cell suspensions prepared from the spleen. The primary immune response to SRBC was determined using the splenocyte direct PFC assay as described (Smialowicz et al., 1991). The expression of splenic lymphocyte surface markers from MXC-dosed and SRBC-immunized rats was measured by multiparameter flow cytometry as described (Smialowicz et al. (1994). The monoclonal antibodies employed were fluorescein isothiocyanate (FITC)-conjugated rabbit anti-rat W3/25 (CD4, Sera-Lab, Accurate Chemical and Scientific Corp., Westbury, NY), phycoerythrin (PE)-conjugated mouse anti-rat OX8 (CD8, PharMingen, San Diego, CA), and FITC-conjugated rabbit anti-rat IgM (Zymed Laboratories, Inc., San Francisco, CA). All antibodies were pretitrated for optimal fluorescence prior to staining of cells. For dual staining of CD4 and CD8, cells were incubated simultaneously with FITC-anti-W3/25 and PE-anti-OX8. Cells were analyzed using an EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL) equipped with an argon-ion laser operated at a wavelength of 488 nm. Green fluorescence from FITC emission was measured using a 525-nm band-pass interference filter, and yellow fluorescence from PE emission was measured using a 575-nm band-pass interference filter. A 550 dichroic filter was used to separate the PE and FITC signals. Dead cells and debris were excluded by collecting lymphocyte population data gated on forward and 907 light scatter.

Group G: Immunotoxicity Assessments

Groups H and I: Reproductive Toxicity Assessments

The remaining animals were dosed daily until pnd42. At this time, six each males and females per dose level were transferred to the NHEERL laboratories and allowed to mature for immunotoxicologic evaluation. Eight-week-old male and 10-week-old female rats were killed by CO2 asphyxiation and the spleen, thymus, and mesenteric lymph nodes were aseptically removed. Terminal body, spleen, and thymus weights were recorded. Lymphoproliferative response and natural killer cell activity. The lymphoproliferative response (LPR) of splenic lymphocytes, cultured for 72 h at 377C and 5% CO2, was determined by measuring [3H]thymidine incorporation as described by Smialowicz et al. (1989). The mitogens em-

Like the animals for immunotoxicity assessment and those subject to necropsy at puberty, these animals were evaluated during maturation for day of vaginal opening and preputial separation. After the end of dosing and after vaginal opening, females were subject to daily vaginal lavage to evaluate estrous cyclicity by cytology. In this study, lavage was performed daily between pnd42 and mating at ca. 12 weeks of age. During this period, animals were weighed weekly. Group H was used to examine functional and structural alterations in the reproductive systems of adults. One male and one female from each litter were mated: each treated male to two untreated females, and every two

Group F: Pubertal Necropsy

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

ftoxal

142

CHAPIN ET AL.

treated females to one proven untreated male. The resulting litters were delivered, and the pups counted, sexed, weighed, and remained with the dam until pnd10 to assess lactational sufficiency (measured as pup weight gain and survival). The pups were removed, killed, and discarded pnd11, and the dams remated with a different partner after 3–7 days. For both matings, the males and females were separated when sperm was detected in the daily vaginal lavage or after 7 days of mating, whichever occurred first. In the second mating, the dams were killed 2 days prior to delivery (gd19, when these females were É165 days old) , and uterine contents were evaluated for fetal number, sex, and number and position of dead implants and resorptions. Corpora lutea were counted macroscopically and the ovaries fixed in Bouin’s. The remaining organs were examined for visible structural malformations, and selected tissues (liver, kidneys, adrenals, thymus, and spleen) were removed, weighed, and fixed in 10% neutral buffered formalin. Nonpregnant females underwent similar necropsy procedures; uteri were examined by transillumination in a Plexiglass press-plate for evidence of occult implantations. At termination, treated males were anesthetized with 30% O2/70%CO2 at Épnd152, and two samples of blood collected from the retroorbital sinus into K/-EDTA and nonheparinized tubes, respectively, for hematology and clinical chemistries. These were examined in males to avoid the possible confounding effects of pregnancy. For clinical chemistry, alanine aminotransferase, lactate dehydrogenase, total protein, glucose, sodium, potassium, and chloride were measured using reagents from Instrumentation Laboratory (Lexington, MA). Alkaline phosphatase, sorbitol dehydrogenase, 5*-nucleotidase, albumin, urea nitrogen, and magnesium were measured with reagents from Sigma. All clinical chemistry endpoints were evaluated on a IL Monarch 2000 chemistry system with ISE (Instrumentation Laboratory) using protocols supplied by the manufacturer. Blood samples treated with EDTA were assayed using the Technicon H*1 hematology analyzer (Bayer, Inc., Tarrytown, NY). A complete blood count (CBC) was performed following manufacturer’s protocols. A spun microhematocrit was measured for comparison with the automated value. The H*1 analyzer provided a differential count of the WBCs by sorting the cells according to peroxidase activity. Wright-Giemsa stained blood smears were prepared for examination of cellular morphology and for verification of any abnormal automated platelet or WBC differential values. Reticulocyte counts were determined by mixing equal amounts (50 ml) of whole blood and New Methylene blue stain and allowing to incubate at room temperature for 15 min. Smears were prepared and the percentage value was calculated from the number of reticulocytes per 1000 RBCs. Animals were then killed by asphyxiation with CO2 . The gonads and associated reproductive organs (seminal vesicles, coagulating gland, prostate (all lobes)) were examined carefully for structural malformations, weighed, and processed. The left testis was frozen for measure of testicular spermatid head count (Robb et al., 1978), right testis and epididymis were placed in Bouin’s fixative, embedded in paraffin, and stained with periodic acid and Schiff’s stain, counterstained with hematoxylin. The left cauda epididymis was incised to collect sperm for determining sperm motility (method in Harris et al., 1992) and then chopped, incubated for 15 min in 10 ml of MEM at 377C, and fixed with 500 ml of 50% glutaraldehyde for sperm counting using a hemacytometer. Fixed tissues from all animals in Group H were embedded in paraffin, 5-mm sections were stained with eosin and hematoxylin (PAS-H for testes), and evaluated by light microscopy without knowledge of treatment group. Numbers of primordial and small follicles in the control and 150MXC groups were counted without knowledge of treatment group in 12–15 step sections of both ovaries in five rats per treatment group. The use of Group I females is triggered if the analysis of Group H data finds that fewer treated females were ovulating or mating. This was indeed the case in this study; no high-dose females ever became pregnant. The possibility of abnormal hormonal control of ovulation was assessed with the Group I females, who were subject to vaginal lavage and (intended n Å 10) killed on the afternoon of proestrus, or the morning of estrus according to vaginal cytology, or after 10 days if the rat was not cycling. Immediately

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

prior to death, blood was collected by cardiac puncture under light CO2 anesthesia. Serum estradiol and progesterone were measured using radioimmunoassay kits from Diagnostic Products, Inc. (Los Angeles, CA); follicle stimulating hormone (FSH) was measured using kits from Amersham Corp., (Arlington Heights, IL). The interassay and intraassay coefficients of variation of the assays were less than 10%. Dose-Range Finding (DRF) A dose-range finding study was performed with two to three dams/treatment level and six levels of exposure and controls. The animals were dosed as above (dams dosed gd14 through pnd7 and then pups directly dosed pnd7–pnd42). Endpoints included viability, body weight gain, day of vaginal opening, prepuce separation, terminal body weight, gross lesions (none observed), organ weights, and histology. Dose levels for the DRF study were 0, 20, 50, 100, 130, 170, and 200 mg/kg/day. Data from this study were used to set doses for the main study at 0, 5, 50, and 150 mg/kg. Statistical Analysis Neurobehavioral evaluations. Individual neurobehavioral measures were analyzed as described by Creason (1989). Two-way ANOVAs were conducted with treatment as a grouping factor and repeated testing as a within-subject factor. Continuous data were analyzed by a linear model (GLM; SAS, 1990), while rank data were subjected to a categorical analysis procedures (CATMOD; SAS, 1990). If the overall dose-by-time interaction in the ANOVA was significant at p õ 0.05, then the data from each time point were analyzed using a one-way ANOVA, followed by post hoc tests to determine which dose groups were significantly different from control. A significant overall dose factor prompted further analyses with the data collapsed across time to determine significant dose groups. Crossing latencies in the training trial, at 24 h, and at 2 weeks were the dependent variables from the passive avoidance experiments subjected to analysis. For each test day, latency values were compared across treatment groups using a Kruskal–Wallis analysis for non-parametric data. Immunology data. Data were analyzed as previously reported (Smialowicz et al., 1994). Means were considered significantly different at p ° 0.05. Reproductive data. Linear regression was used to assess dose–response trends (Neter et al., 1985). Pairwise comparisons (treated groups vs controls) were made using Dunnett’s test (Dunnett, 1955). Pups for these studies were generated in two replicate dosings, or cohorts. When data from more than one study cohort were available (dams’ body weights and litter counts) and pups through pnd46 (body weights, organ weights, AGD measurements, pnd of vaginal opening/preputial separation) two-way analysis of variance methods were used to ascertain statistically significant differences between dose groups and study cohort. When data were collected from only one study cohort, one-way analysis of variance methods were used to determine if there were significant differences between the dose groups. Nested analysis of variance methods were used to determine if significant litter effects existed in the offspring (neonate and weanling) endpoints. Pairwise results were not reported unless there was a significant (or borderline significant) trend or difference between doses (the same is true for the other multiple comparison procedures mentioned later in this section). Less than half of the endpoints included in the text and tables had significant study group effects and very few had a significant dose-by-study group effect (or both). Even though significant differences between the two study groups did exist, most of the effected endpoints had consistent dose effects within each study group. Therefore, the tables represent the pooled study cohort data for these endpoints. (Note that the study cohort 2 females did not have any control or high dose weanlings with terminal body and organ weights (pnd46) and the study cohort 1 males did not have any high dose weanlings with terminal body and organ weights (pnd46) or any middle or high dosed weanlings with a preputial separation pnd.) Significant litter effects existed for all body weight and ano-genital distance endpoints through pnd42 and for some of the terminal body and organ weights col-

ftoxal

143

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE lected by pnd46. Day of vaginal opening/preputial separation also revealed significant correlation of littermates. Accordingly, the data for these endpoints were averaged over litter and analyzed statistically using the litter averages. Due to the maturing of these offspring and decreasing sample sizes of the original litters, data collected for endpoints from adulthood (after pnd46) were analyzed for litter effects but had little power to detect ‘‘true’’ statistical significance. Therefore, these endpoints were not averaged over litter. Two treated males (one control, one low dose) were removed from the clinical chemistry data prior to analysis after the Dixon and Massey outlier test indicated that 4 of 14 endpoints were statistical outliers. For a third male in the middle dose group a single data point was removed prior to analysis of only one endpoint for hematology data. The Freeman–Tukey transformation for count data (Anscombe, 1948) (sqrt(n)/sqrt (n / 1)) was applied to most of the count endpoints, the arcsine square-root transformation for proportions (arcsine(sqrt(proportion))) was applied to percent (sperm) motile, and the square root transformation was applied to platelet counts (treated males). Transformations were applied prior to statistical analysis. The tables present the means and standard errors of the untransformed data. Adjusted pup weights were tested for dose effects using analysis of covariance methods, using litter size as the covariate. Means (and standard errors) in the tables represent the least squares estimates of mean (and standard error) pup weight, adjusted for litter size. T tests of differences in least squares means were used as the multiple comparison procedure. Male fertility and pregnancy rates in the mating trials were assessed for dose–response using the Cochran–Armitage test for a linear trend in proportions (Armitage, 1971), followed by a one-sided Fisher’s exact test as the multiple comparison procedure. Male fertility for the treated males was analyzed for each mating trial separately. Pregnancy rates for both the treated and the untreated females were also analyzed for each mating trial separately. Adjusted pup weights on pnd1 and 7 were calculated on all pups but the unadjusted pup weights on both of these days and the AGD measurement on pnd1 were calculated on only the pups that were left after standardization on pnd7 (Table 2). One control dam from the first replicate produced a litter of size 2, which influenced the least squares analysis of covariance for the adjusted pup weights. Accordingly, this dam was excluded from the adjusted pup weight analysis. The number dead/litter counts (dams and in the mating trials) represent the number of pups that died since the last day analyzed. In the second mating of the mating trials (Tables 5 and 7), a female was considered pregnant if her number of implants was greater than zero. Corrected maternal weight (gd19) for treated pregnant females was calculated: (gd19 body weight) 0 (gravid uterus weight) / (empty uterus weight). Second mating endpoints were calculated on the pregnant females only except for male fertility, pregnancy rates, and number of corpora lutea counts (included only nonzero values). Two untreated females in the second mating (one mated to a control male, one mated to a middle dosed male) had corpora lutea counts õ total implant counts. Accordingly, both females were excluded from the analysis of the following endpoints: preimplantation loss (number of corpora lutea 0 total number of implants), total implants/ corpora lutea (%), and number live implants/corpora lutea (%). A control male paired only once in each round of mating was excluded from the male fertility analysis (a pregnancy resulted from the first mating only). For the treated male hematology data a few platelet counts per dose group were excluded prior to the analysis of platelet count endpoint because these samples had clumped platelets. For hormone analysis, animals were grouped according to cycle stage by vaginal histology: vaginal epithelium of proestrus rats had cuboidal epithelial cells overlying cornified squamous epithelium; vaginal epithelium of estrus rats had cornified squamous epithelium. Although rats treated with 50 or 150 mg/kg methoxychlor were not cycling, their vaginal histology was characterized as cornified epithelium consistent with estrogenic or ‘‘estrus’’ histology and were consequently compared to the controls and 5 mg/kg estrus hormone groups. Treatment effects on hormones were assessed using analysis of variance (ANOVA) methods and least significant difference

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

(JMPSAS Institute Inc., Cary, NC). Hormones were normalized by logarithmic transformation prior to applying ANOVA. For all statistical tests, alpha was set at p õ 0.05.

RESULTS

To facilitate reading, animals treated with 5 mg MXC/kg/ day are referred to as ‘‘5MXC,’’ those animals treated with 50 mg MXC/kg/day are referred to as ‘‘50MXC,’’ etc. All data are presented as means { SEM, with the number of animals in parentheses. Means in tables or the text that are statistically different from control are indicated by an adjacent asterisk (*). Because of the quantity of data generated by this study, this section will focus primarily on endpoints that showed dose-related differences from controls. Dam effects. The weight gain for the last week of pregnancy of dams treated with 5MXC was not different from controls, although weight gain for dams treated with 50 and 150MXC was reduced by *17 and *37%, respectively (n Å 29–33). This can be plausibly attributed, at least in part, to the smaller litters these dams were carrying. Postnatal dam weight gain was unaffected by MXC treatment (not shown). The results of chemical analysis of plasma and milk are shown in Table 1. The amount of protein and lactose in dam’s milk on pnd7 was unaffected by MXC, although in the 150MXC group, the amount of triglycerides and total lipids as a volume percentage of the milk were both increased. These changes are not viewed as adverse until it is shown that that there will be additional functional consequences on the offspring. Although not quantified, milk was harder to obtain from the treated dams, and generally less milk was obtained. MXC and the monohydroxy and dihydroxy metabolites were present in dam plasma at levels roughly consonant with dose level (Table 1). Statistics were not performed on these chemical distribution data. Milk levels had an apparently steeper dose relationship: at the low dose, MXC was found in milk at levels less than dam plasma, while higher dose levels produced milk MXC levels that exceeded dam plasma MXC levels. Pup plasma levels were detectable only for MXC and for the metabolites at the upper two dose levels. The difference between levels in dams and pups could, in part, be explained by the logistical necessity that pup plasma was sampled É27–30 h after the previous dam’s dosing, while dam milk and plasma were sampled 3–6 h after dosing. This is logical speculation, because the half-life of these metabolites has not been determined in neonates. Monohydroxy-methoxychlor olefin was below the limit of quantitation in milk and pup plasma. Pup development. Pups from Groups C, D, E, F, G, H, and I were evaluated during development (Table 2). Fewer live pups were born at 150MXC. Pup birth weight (both absolute and adjusted for litter size) was decreased by É10– 20% at 150MXC, but was unchanged at lower doses. AGD

ftoxal

144

CHAPIN ET AL.

TABLE 1 Chemical Analyses of Milk and Plasma, PND 7 Methoxychlor (mg/kg/day) 0 Milk Total protein, g/dl Lactose, g/dl Triglycerides, g/dl % Lipids Methoxychlor (MXC), ng/ml Dam plasma Dam milk Pup plasma Monohydroxy-MXC, ng/ml Dam plasma Dam milk Pup plasma Dihydroxy-MXC, ng/ml Dam plasma Dam milk Pup plasma Monohydroxy-MXC olefin, ng/ml Dam plasma

9.4 21.6 7.2 11.7

{ { { {

0.2 (12) 1.4 0.7 0.9

5

9.2 21.5 6.7 12.5

{ { { {

0.2 (9) 1.4 0.6 0.5

50

9.4 23.0 7.0 11.9

{ { { {

0.2 (8) 0.6 0.6 0.4

150

9.2 18.1 10.1 14.8

{ { { {

0.2 (8) 1.6 0.6* 0.6*

19.3 { 4.7 (7) õ50 ng/ml õ5 ng/ml

360.1 { 177 (9) 89.0 { 7.8 (9) 12.4 (1)

2205.0 { 600 (8) 2823.0 { 1216 (8) 37.8 { 3.7 (9)

3938.0 { 701 (8) 8695.0 { 1038 (8) 59.9 { 4.8 (8)

19.6 { 2.8 (9) õ50 ng/ml õ5 ng/ml

36.7 { 4.0 (9) õ50 ng/ml õ5 ng/ml

210.7 { 38 (8) 255.7 { 89 (7) õ5 ng/ml

466.2 { 104 (8) 1004.0 { 162 (8) 6.2 (1)

9.5 { 3.0 (4) õ50 ng/ml õ5 ng/ml

12.4 { 3.6 (8) õ50 ng/ml õ5 ng/ml

108.9 { 23 (6) 125.8 { 42 (6) 6.4 { 0.5 (5)

352.9 { 50 (8) 342.9 { 24 (8) 11.5 { 1.3 (8)

23.8 { 4.7 (6)

45.0 { 14.8 (9)

31.1 { 11.3 (7)

34.0 { 8.8 (8)

Note. Data are mean { SEM (n). Limits of quantitation were 50 or 5 ng/ml, as indicated. * p õ 0.05 compared to the controls.

on pnd1 was unaffected in either sex by MXC exposure (Table 2). However, vaginal opening (VO) was accelerated in all MXC groups: in controls, the mean age at VO was 37.4 days, while it was reduced by *2, *7, and *4 days in the low to high MXC groups, respectively. Additionally, the mean day of prepuce separation was delayed in the 50MXC and 150 MXC males by É*8 and *34 days, respectively (Table 2). Neurotoxicity assessment. There were no differences among treatment groups on the passive avoidance task. There were, however, some statistically significant alterations obtained in some of the neurobehavioral measures. Most of the analyses showed an overall dose effect but no interaction between dose and time, indicating that the group differences were stable across time. Male rats treated with the highest dose (150MXC) displayed clearly increased handling reactivity throughout testing, but this was the only indication of increased excitability (i.e., related endpoints such as arousal or other activity measures were not affected). There were statistically significant but non-dose-related changes in locomotor activity (increased in females at 50MXC), click response (decreased in 5MXC females, decreased in males at 5MXC and 150MXC), and approach stimulus (lower in 5MXC males). A few measures showed a statistically significant dose-bytime interaction, indicating that the effect of treatment was not consistent over testing. Urination in the open field was increased in female rats in a dose-dependent manner (50MXC

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

and 150MXC) on the last day of testing only (pnd66). Defecation also revealed a significant dose-by-time interaction in female rats, but no one-way ANOVAs for each test day were significant; however, there appeared to be more defecation at 150MXC on day 31. Lower hindlimb grip strength emerged over time in the 50MXC female rats (*80 and *82% of control values on pnd47 and 66, respectively). Pubertal necropsy. Extra animals from both cohorts were killed by asphyxiation and necropsied on pnd46, 4 days after the end of dosing. Because of the animal requirements for other parts of the study and unanticipated reduction in live births in the high dose group, there were fewer than the desired number of high dose pups. The available data are presented in Tables 3 and 4. Body weights for both male and female pups in the high dose group were reduced occasionally during growth to pnd46, by which time the differences were slight and nonsignificant. Thymus weight was decreased in the middle-dose males by É*15% and in both sexes of high dose pups by É*30%. Weights of testis, epididymis, seminal vesicles, and prostate were all reduced in the middle dose group by É*30% and in the high dose group by *43–70%. Uterus weight was reduced at the high dose by *35%, while ovarian weight was reduced in the low, middle, and high dose groups by É*30%, *50%, and *50%, respectively (Table 4). Histologic observations. Testes from the control males contained all steps and stages of spermatogenesis, although the numbers of steps 18 and 19 spermatids were clearly less

ftoxal

145

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE

TABLE 2 Neonate and Pup Data during Maternal and Pup Exposure to Methoxychlor Methoxychlor (mg/kg/day)

No. of Litters Neonates PND 0 Live/litter Dead/litter PND 1 Live/litter PND 7 Live/litter Live/litter following culling PND 21 Live/litter Dead/litter Average Body Weight (g) Absolute PND 1 PND 7 PND 14 PND 21 Adjusted PND 1 PND 7 Female AGD PND 1 (mm) AGD/body weight PND of vaginal opening Male AGD PND 1 (mm) AGD/body weight PND of preputial separation

0

5

50

150

29

32

33

29

10.9 { 0.6a 0.1 { 0.06

10.7 { 0.4 0.1 { 0.04

11.0 { 0.4 0.2 { 0.15

9.1 { 0.5* 0.9 { 0.20*

10.9 { 0.6

10.5 { 0.5

10.8 { 0.5

8.7 { 0.6*

10.9 { 0.6 (29) 7.3 { 0.2

10.5 { 0.4 (31) 7.3 { 0.2

11.2 { 0.4 (32) 7.0 { 0.3

9.3 { 0.5 (29) 6.8 { 0.4

7.3 { 0.2 0

7.5 15.5 32.4 51.8

{ { { {

0.2 0.5 0.8 1.2

7.3 { 0.2 0

7.3 15.7 32.4 51.7

{ { { {

0.1 0.5 0.9 1.4

7.0 { 0.3 0

7.2 14.4 30.7 50.6

{ { { {

0.1 0.4 0.5 0.9

6.3 { 0.5 0.4 { 0.4

6.9 13.6 27.2 46.3

{ { { {

0.2 0.6* 0.8* 1.6*

7.6 { 0.1 15.7 { 0.3

7.2 { 0.1* 15.8 { 0.3

7.3 { 0.1 14.6 { 0.3*

6.6 { 0.1* 12.5 { 0.3*

1.8 { 0.1 0.25 { 0.01 37.4 { 0.6

1.7 { 0.1 0.25 { 0.01 35.2 { 0.5*

1.7 { 0.1 0.25 { 0.01 30.8 { 0.2*

1.7 { 0.1 0.25 { 0.02 33.4 { 0.3*

2.9 { 0.08 0.38 { 0.01 42.6 { 0.5

2.9 { 0.1 0.39 { 0.01 42.1 { 0.4

2.9 { 0.1 0.40 { 0.01 50.5 { 1.4*

2.9 { 0.1 0.41 { 0.01 76.5 { 3.4*

Note. PND, postnatal day; AGD, anogenital distance. a Mean { SEM (N) where N Å number live litters. * p õ 0.05 compared to controls.

than found in adults. As dose of MXC increased, testes of treated animals showed progressive inhibition of development. Cell death was not prominent, but fewer mature germ cells were present, and fewer germ cells of each type were present. The interstitium appeared normal. The accessory sex organs appeared structurally normal, although smaller. Ovaries of control and 5MXC were morphologically comparable and characterized by an admixture of follicles of all sizes, mature corpora lutea, and interstitial tissue. Ovaries from the 50 MXC and 150MXC were hypoplastic and characterized by a predominance of primordial and small follicles, few antral follicles and an absence of corpora lutea (Figs. 2–4). Uteri from controls and 5MXC rats were normal, and indistinguishable. Two of seven uteri from 50MXC rats showed mild to marked hyperplastic growth; four of five uteri at 150MXC showed mild to marked endometrial hyperplasia (Fig. 5) and one showed squamous metaplasia (Fig. 5).

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

These data showed that MXC had demonstrable adverse effects on the development of the male and female reproductive systems, stimulating vaginal opening while inhibiting ovarian development. In males, reproductive development was generally delayed, in some cases to a substantial degree (preputial separation). Adult immunotoxicity assessments. No dose-related or significant changes were observed for the following endpoints: spleen weight, splenic natural killer cell activity, splenic lymphoproliferative response, female antibody plaque-forming cell number, or splenic cell-surface phenotypes. On the other hand, absolute and relative thymus weights were reduced in the pnd46 males at 50 and 150MXC (Table 3), and in the 150MXC females at pnd46 and 165 (Tables 4 and 6). There was also a significant decrease in male plaque-forming cells/spleen. The 5MXC and 50MXC males were *35% and *42% less than the control cell number (483 { 46 1 104/spleen, n Å 6). Because of the perinatal

ftoxal

146

CHAPIN ET AL.

TABLE 3 Male Pubertal Necropsy Data (Group F) after Exposure to Methoxychlor Methoxychlor (mg/kg/day)

No. of litters No. of neonates Body weight (g) PND 21 PND 28 PND 35 PND 42 Body weight change (g) PND 21–28 PND 28–35 PND 35–42 PND 46 terminal weight (g) No. of rats Body weight Liver Absolute Relative Kidney Absolute Relative Adrenals Absolute Relative Spleen Absolute Relative Thymus Absolute Relative Testes Absolute Relative Epididymis Absolute Relative Seminal Vesicles Absolute Relative Prostate Absolute Relative

53.0 87.0 135.0 179.0

0

5

50

150

20 60

21 62

22 64

13 40

{ { { {

1.3a 1.8 2.5 3.4

52.0 88.0 137.0 184.0

{ { { {

1.4 2.2 3.0 3.8

52.0 87.0 134.0 175.0

34.2 47.8 45.5

{ 0.8 { 1.2 { 1.4

35.5 49.3 47.5

{ 1.1 { 1.1 { 1.0

35.2 47.0 40.8

203.0

24 { 4.9

208.0

26 { 4.2

193.0

{ { { {

1.0 1.5 1.9 2.7

{ 0.7 { 1.0 { 1.0*

28 { 3.4

47.0 81.0 126.0 157.0 33.7 45.4 34.1

184.0

{ { { {

1.7* 2.7 3.3 3.0*

{ 1.3 { 1.3 { 1.6*

10 { 5.1

9.38 { 0.30 4.61 { 0.05

10.00 { 0.30 4.81 { 0.09

9.22 { 0.20 4.78 { 0.07

9.59 { 0.30 5.21 { 0.10*

1.64 { 0.05 0.81 { 0.01

1.70 { 0.05 0.82 { 0.02

1.57 { 0.04 0.82 { 0.01

1.73 { 0.05 0.94 { 0.03*

0.028 { 0.002 0.014 { 0.001

0.034 { 0.003 0.017 { 0.001

0.031 { 0.001 0.016 { 0.001

0.038 { 0.003* 0.021 { 0.002*

0.66 { 0.03 0.33 { 0.01

0.68 { 0.04 0.33 { 0.02

0.65 { 0.03 0.34 { 0.01

0.75 { 0.02 0.41 { 0.01*

0.69 { 0.02 0.34 { 0.01

0.69 { 0.02 0.33 { 0.01

0.58 { 0.02* 0.30 { 0.01*

0.47 { 0.02* 0.25 { 0.01*

2.17 { 0.05 1.07 { 0.01

2.09 { 0.05 1.00 { 0.02

1.54 { 0.07* 0.80 { 0.03*

0.61 { 0.06* 0.33 { 0.02*

0.28 { 0.01 0.14 { 0.01

0.28 { 0.01 0.13 { 0.003

0.22 { 0.01* 0.11 { 0.01*

0.12 { 0.003* 0.07 { 0.001*

0.130 { 0.010 0.064 { 0.004

0.151 { 0.010 0.073 { 0.004

0.089 { 0.010* 0.047 { 0.007

0.066 { 0.010* 0.036 { 0.007*

0.078 { 0.007 0.038 { 0.003

0.080 { 0.005 0.038 { 0.002

0.053 { 0.004* 0.027 { 0.002*

0.039 { 0.005* 0.021 { 0.003*

a

Mean { SEM. * p õ 0.05 compared to controls.

pup death, there were insufficient animals at the high dose to contribute to all endpoints, and no 150MXC males were evaluated for this endpoint. Reproductive toxicity assessments. Estrous cyclicity was monitored during growth to mating in the 15 females per group. In the 4 weeks immediately prior to mating, the proportion of animals who had normal cycles in the control, low, middle, and high dose groups were 13/15, 14/ 15, 3/15, and 2/15, respectively. Cycles were highly irregular or absent at 50MXC and 150MXC: 11 females in each

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

group had extended (ú3 days) durations of estrous or proestrous smears. Finally, smears showing cells characteristic of extended (ú3 days) diestrous were seen in 2, 1, 1, and 2 females in the control to high dose groups, respectively. Thus, the control and low dose groups showed similar patterns of smears, while the middle and high dose groups both showed irregular and disrupted vaginal cycle patterns. In the mating trials, 15 males were each cohabited with two naive, untreated females for 7 days, or until sperm were

ftoxal

147

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE

TABLE 4 Female Pubertal Necropsy Data (Group F) after Exposure to Methoxychlor Methoxychlor (mg/kg/day)

No. of litters No. of neonates Body weight (g) PND 21 PND 28 PND 35 PND 42 Body weight change (g) PND 21–28 PND 28–35 PND 35–42 PND 46 terminal weight (g) Number of rats Body weight Liver Absolute Relative Kidney Absolute Relative Adrenals Absolute Relative Spleen Absolute Relative Thymus Absolute Relative Ovaries Absolute Relative Uterus Absolute Relative

51.0 82.0 123.0 157.0

0

5

50

150

19 56

21 71

20 63

17 52

{ { { {

1.3a 1.8 2.3 2.7

51.0 81.0 121.0 153.0

{ { { {

1.5 1.7 2.2 2.2

50.0 80.0 120.0 151.0

31.1 40.3 34.1

{ 0.8 { 0.8 { 0.8

30.4 40.3 31.3

{ 0.6 { 0.7 { 0.7

30.7 39.3 31.1

180.0

11 { 3.6

168.0

23 { 2.6

162.0

{ { { {

1.1 1.9 2.5 3.1

{ 1.0 { 0.7 { 0.9*

15 { 8.6

46.0 77.0 117.0 146.0

{ { { {

1.3* 2.2 2.6 2.7*

30.5 40.1 29.4

{ 1.1 { 0.9 { 0.8*

160.0

8 { 5.4

8.54 { 0.20 4.74 { 0.06

7.71 { 0.10 4.58 { 0.04

7.92 { 0.50 4.87 { 0.09

8.02 { 0.40 4.99 { 0.10

1.45 { 0.04 0.81 { 0.02

1.39 { 0.02 0.83 { 0.01

1.38 { 0.07 0.86 { 0.02

1.36 { 0.07 0.85 { 0.02

0.036 { 0.003 0.020 { 0.001

0.034 { 0.002 0.021 { 0.001

0.036 { 0.002 0.022 { 0.001

0.035 { 0.003 0.022 { 0.002

0.55 { 0.03 0.31 { 0.01

0.52 { 0.02 0.31 { 0.01

0.53 { 0.04 0.33 { 0.01

0.56 { 0.03 0.35 { 0.01

0.61 { 0.03 0.34 { 0.02

0.57 { 0.02 0.34 { 0.01

0.52 { 0.04 0.32 { 0.02

0.43 { 0.02* 0.27 { 0.01*

0.065 { 0.004 0.036 { 0.002

0.047 { 0.003* 0.028 { 0.002

0.030 { 0.043* 0.019 { 0.003*

0.03 { 0.008* 0.02 { 0.004*

0.26 { 0.02 0.15 { 0.01

0.27 { 0.02 0.16 { 0.01

0.23 { 0.02 0.14 { 0.01

0.17 { 0.02* 0.11 { 0.01

a

Mean { SEM. * p õ 0.05 compared to controls.

present in the vaginal lavage fluid, starting at ca. 12 weeks pairs per group, 10, 11, *2, and *0 females became pregnant of age. Females were partnered with breeder-certified proven in the control to high dose level, respectively (Table 5). Dams males. were killed immediately prior to parturition. The 50MXC Females. In the first female mating trial, of the 15 cohab- litters had only *32% of the implants found in the controls. ited females/group, 13, 11, *3, and *0 females became preg- There was no increase in uterine resorptions, and the corpora nant in the controls and low to high dose groups, respectively lutea count was significantly reduced in the 50MXC females, (Table 5). At the high dose, no pairs mated, and no litters leading to an increase in the calculated preimplantation loss were delivered. In those animals delivering, there was no probably mediated by reduced ovulation. Organ weight data from these pregnant females are prestatistically significant change in litter size; although litter sented in Table 6. Female body weights were not different size was clearly decreased at 50MXC (8.3 pups/litter, vs across groups, nor were absolute or adjusted weights of liver, 11.7 in control); the lack of statistical significance is likely kidney, or spleen. Thymus weights were increased at due to the low number of litters (n Å 3). There were no treatment-related effects on pup mortality or weight gain to 50MXC (by 60%, n Å 2), and there was a monotonic reduction in uterine weight in pregnant animals that was signifipnd10, when the pups were removed and killed. In the second mating of treated females, of the 15 breeding cant for both the 5MXC and 50MXC groups.

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

ftoxal

148

CHAPIN ET AL.

FIG. 2. Photomicrograph of an ovary from 42-day-old control rat. Arrows point to corpora lutea; arrowheads point to developing follicles of all stages. Bar represents 250 mm.

In the nonpregnant females also euthanized at this time, there was a monotonic increase in pyelonephritis. There were no changes in the weight of liver, kidney, or spleen; thymus weight was decreased by É30% at 150MXC, and uterus weight was the same across groups (Table 6). Histologically, there were no significant microscopic lesions in female livers, kidneys, thymus, or spleens. The microscopic structure of all tissues evaluated in the control and low dose Group H females (uterus, vagina, ovary, mammary gland) was indistinguishable from that of controls; no treatment effects were detected. Most females showed tissue changes characteristic of pregnancy, consistent with their death near term. A single 5MXC female had ovarian cysts; no ovarian cysts were noted in controls. At 50MXC, only two animals had uterine implants. Uteri from the remaining animals had mild to severe endometrial squamous metaplasia and endometrial hyperplasia. Vaginas

FIG. 4. Photomicrograph of an ovary from 42-day-old treated with 150 MXC mg/kg/day. This ovary is typical of the 50- and 150 MXC groups, and contains large antral and early cystic follicles (asterisks) in addition to numerous small follicles, but a paucity of corpora lutea.

from six animals at 50MXC were cornified, an estrogenic response. One female had vaginal epithelial hyperplasia. Most (11/13) ovaries in this group had ú4 fluid-filled cysts (were polycystic), while two females had cystic oviducts, and one had hypertrophy of the oviduct muscle layer. While the mammary tissue of two females contained alveoli, mammary tissue of seven others contained no visible alveoli; only ducts were visible. At 150MXC, no animals were pregnant. Eleven of 15 females had uterine squamous metaplasia, while 8/15 had endometrial hyperplasia. Vaginas of 8 females were cornified, and 1 showed epithelial hyperplasia. One polycystic ovary was observed, but ovaries from most (11) females were atrophied, with little or no detectable follicular development and few/no corpora lutea (Fig. 6). There were 5 cystic oviducts, and oviductal muscular hypertrophy in 2

FIG. 3. (A) Photomicrograph of an ovary from 42-day-old treated with 150 MXC g/kg/day. Arrows point to large area of grouped primordial and small follicles; rest of ovary contains numerous antral follicles. Note the paucity of corpora lutea. Bar represents 250 mm. (B) Higher magnification of A to demonstrate the groups of numerous primordial and small follicles.

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

ftoxal

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE

149

FIG. 5. (A) Photomicrograph of uterus from 42-day-old control rat. (B and C) Photomicrographs of uterus from 42-day-old rat treated with 150 mg MXC/kg/ day characterized by epithelial hyperplasia (B) and squamous metaplasia (arrows in C).

females. Mammary tissue from 11 females contained only ductal tissue, with no alveolar development. Males. Each male (n Å 15/group) was cohabited with two nontreated females. The first litter was delivered, reared to pnd10, and then removed and the animals randomly paired to produce another pregnancy. The pregnant dams were killed immediately prior to necropsy, and uterine contents examined. The male mating trials data are shown in Table 7. The proportion of females with vaginal sperm was reduced at 150MXC in both mating trials. The size of the resulting litters was not reduced, and there was no effect of paternal MXC exposure on postpartum pup deaths or weight gain. In the second mating trial where the females were killed preterm, the number of uterine implants was not reduced by paternal MXC exposure, nor were there increases in resorptions or preimplantation loss (Table 7). The males were euthanized and necropsied (Table 8). There was no treatment-related reduction in body weight, or in the weights of liver, kidneys, spleen, adrenals, or thymus. There were no gross structural abnormalities in the male reproductive tract observable at necropsy. Weights were significantly reduced for testis and right epididymis at 50MXC

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

and 150MXC, while weights of left cauda epididymis, seminal vesicles, and ventral prostate were significantly reduced at 150MXC. The proportion of motile sperm was reduced by *14% at 150MXC, while epididymal sperm density and the number of testicular homogenization-resistant spermatid nuclei per testis were each reduced by *33%. Sperm counts were unchanged at 50MXC and 5MXC. No treatment-related changes were detected in clinical chemistries and hematology (not shown). Histologically, there were no significant microscopic lesions in male liver, kidney, prostate, seminal vesicles, thymus, or spleen. Of 15 animals/group whose testes were evaluated (representing 9–12 litters/group), minimal epithelial disorganization (defined as reduced numbers of germ cells, germ cells displaced from their normal position within the epithelium, the presence of abnormally large amounts of Sertoli cell cytoplasm visible, and/or marked reductions in numbers of elongated spermatids) was present in no controls, 1 rat at 5MXC and 2 rats at 50MXC, while mild disorganization was present in 1 rat each at 50MXC and 150MXC. The testes of one rat at the top dose were completely atrophic; the seminiferous tubules consisted of Sertoli cells and sper-

ftoxal

150

CHAPIN ET AL.

TABLE 5 Female Mating Trial Data in Adults (Group H) after Neonatal/Juvenile Methoxychlor Exposure Methoxychlor (mg/kg/day)

First mating trial Females pregnant/cohabited Neonates PND 0 No. live litters Live pups/litter Dead pups/litter PND 1 No. live litters Live pups/litter Dead pups/litter PND 10 No. live litters Live pups/litter Dead pups/litter Second mating trial Females pregnant/cohabited Mean estrous cycle length (days) Mean live fetuses/dam Mean resorptions/dam Mean implants/dam Mean corpora lutea/dam PreImplantation loss

0

5

50

150

13/15

11/15

3/15*

0/15*

13 11.7 { 0.6 0.1 { 0.1

11 11.6 { 1.1 0.2 { 0.2

3 8.3 { 4.3 0.3 { 0.3

18 11.7 { 0.6 0.0

11 11.2 { 1.2 0.5 { 0.2*

2 12.5 { 1.5 0.3 { 0.3

13 11.4 { 0.6 0.0

10 11.7 { 0.5 0.05 { 0.05

2 12.0 { 1.0 0.0

10/15 5.2 { 0.3 13.8 { 1.1 1.4 { 1.0 15.2 { 0.6 21.6 { 2.1 4.5 { 1.1

5.3 11.5 1.1 12.6 17.6 3.3

11/15 { 0.7 { 0.6 { 0.3 { 0.7 { 1.9 { 0.6

2/15* 7.1 { 0.6* 4.5 { 2.5* 0.5 { 0.5 5.0 { 3.0* 13.2 { 2.8* 11.5 { 1.5*

0/15* 6.1 { 0.4

* p õ 0.05 compared to controls.

matogonia only. In the 150MXC rat with mild disorganization, É12% of tubules showed very few postspermatogonial germ cells and much visible Sertoli cell cytoplasm. This presentation of affected tubules interspersed among many normal-appearing tubules is consistent with a single seminiferous tubule being affected throughout its length, while the other seven tubules of the testis were normal. Hormone females. In the 5MXC females, the estradiol:progesterone ratio was not different from controls during proestrus (not shown). The serum estrogen:progesterone ratio was significantly elevated in the 50MXC and 150MXC groups compared to control and 5MXC during estrus (Fig. 7). This alteration is attributed to a lack of progesterone due to absence of ovulation and corpora lutea formation at 50 and 150MXC. While serum FSH levels were not different between control and 5MXC rats on proestrous (Fig. 8), FSH levels were significantly suppressed in all MXC-treated rats determined to be in estrus by vaginal histology (Fig. 9). Other tissues were examined microscopically from these females, and the changes were generally the same as the mated females with the following two exceptions: (1) In three 5MXC females, the uteri had mild to moderate areas of focal to locally extensive squamous metaplasia, while ovaries were normal. A single focus of similar metaplasia was seen in one control. (2) The ovaries from the 150MXC

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

females were uniformly polycystic, while the ovaries from the mating group of 150MXC females were atrophic. Ovaries from 50MXC and 150MXC rats had polycystic ovaries and uterine hyperplasia and metaplasia. In 3/20, 17/20, and 16/16 females in the 5MXC, 50MXC, and 150MXC groups, respectively, uteri were composed of hyperplastic endometrium and/or focal or locally extensive areas of squamous epithelium (metaplasia) (not shown). There was no significant difference in total follicle counts between controls (66 { 22) and 150MXC (90 { 32) groups. DISCUSSION

The present experiments found detectable amounts of parent compound and two active metabolites in dam plasma and milk and pup plasma. The data show some apparent nonlinearities with respect to MXC itself: at lower doses, there is more in dam plasma than in milk, but the reverse is true at higher doses. When comparing the pup and dam data, it should be remembered that, because the pups are separated from the dams on the morning of milking and prior to dosing, the dams fluids were collected É4–6 h after dosing, while pups plasma was collected É30 h after their mother’s previous dose. This makes the detectable levels of MXC and the dihydroxy metabolite in pups more significant.

ftoxal

151

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE

TABLE 6 Necropsy Data from Adult Females (Group H) after Neonatal/Juvenile Methoxychlor Exposure Methoxychlor (mg/kg/day) 0 Pregnant female weight (g) GD 19 Body Weight (corrected) n Liver Absolute Relative (b) Kidney Absolute Relative Spleen Absolute Relative Thymus Absolute Relative Empty Uterus Absolute Relative Nonpregnant female weight (g) No. of females No. with abnormalities (ovarian/uterine/renal) Terminal body weight Liver Absolute Relative (b) Kidney Absolute Relative Spleen Absolute Relative Thymus Absolute Relative Empty Uterus Absolute Relative

402.5

{ 6.4a 10

5

392.8

{ 10.5 11

50

405.3

{ 12.1 2

17.90 { 0.58 4.45 { 0.11

16.90 { 0.77 4.34 { 0.14

15.90 { 0.50 3.92 { 0.01

2.26 { 0.07 0.56 { 0.02

2.13 { 0.08 0.55 { 0.02

2.23 { 0.26 0.55 { 0.05

1.33 { 0.16 0.33 { 0.04

1.07 { 0.10 0.28 { 0.03

0.96 { 0.11 0.24 { 0.03

0.19 { 0.02 0.049 { 0.005

0.17 { 0.02 0.046 { 0.005

0.31 { 0.03* 0.081 { 0.01*

5.13 { 0.20 1.33 { 0.06

4.06 { 0.30* 1.07 { 0.06*

2.51 { 0.60* 0.65 { 0.18*

5

4

341.7

0 { 8.1

340.7

1 { 7.3

13

307.3

4 { 10.5

150

0

15 9 324.1 { 11.8

12.20 { 0.40 3.58 { 0.10

11.50 { 0.40 3.38 { 0.07

11.40 { 0.40 3.73 { 0.08

12.00 { 0.50 3.74 { 0.10

2.11 { 0.10 0.62 { 0.04

2.05 { 0.10 0.60 { 0.02

2.16 { 0.06 0.71 { 0.02

2.26 { 0.08 0.70 { 0.02

0.73 { 0.03 0.21 { 0.01

0.69 { 0.02 0.20 { 0.01

0.78 { 0.06 0.25 { 0.01

0.72 { 0.05 0.22 { 0.01

0.22 { 0.02 0.06 { 0.006

0.22 { 0.04 0.06 { 0.011

0.16 { 0.01 0.05 { 0.004

0.14 { 0.01* 0.04 { 0.004*

0.59 { 0.04 0.17 { 0.01

0.54 { 0.15 0.15 { 0.04

0.52 { 0.04 0.19 { 0.03

0.49 { 0.01 0.15 { 0.01

a

Mean { SEM. (Organ weight/corrected dam weight) 1 100. * p õ 0.05 compared to controls. b

These data also extend the observations of Appel and Eroschenko (1992), who reported effects on the reproductive tracts of female pups nursing from mouse dams given doses of 30, 60, and 150 mg/kg MXC. The vaginal opening data are not entirely dose-dependent, as the 150MXC females had a value between the 5 and 50MXC groups. Nonetheless, vaginal opening was accelerated even at 5MXC, the lowest dose tested, while preputial separation was delayed at 50MXC. These doses are lower than the effective doses reported by Gray et al. (1989), which can be explained by the later onset of dosing used by Gray

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

et al. (pnd21) compared to the present design (gd14). While accelerated vaginal opening may not be considered adverse, delayed puberty in the males is adverse. At the least, both can be considered as biomarkers of response to an ‘‘estrogenic’’ stimulus. Reduced hormone levels should probably be considered adverse, inasmuch as normal reproductive function depends on normal hormonal stimulation, and suboptimal stimulation will eventually translate into reduced function, particularly in a population of animals. Profound changes in cognitive function were not expected in this study, and were not seen. The effects that were de-

ftoxal

152

CHAPIN ET AL.

FIG. 6. Photomicrograph of an ovary from 150-day-old rat treated with 150 MXC mg/kg/day. The ovary is larger and contains numerous large antral and cystic follicles (asterisks), few small or developing follicles, and no corpora lutea. Bar represents 250 mm.

tected in these evaluations could suggest changes in the rats’ level of activity/excitability. Male rats showed increased excitability when handled (handling reactivity), and females showed increased locomotor activity. Furthermore, one interpretation of increased urination, as was observed in female rats, is an indication of heightened reactivity (rev. in Archer, 1973). Alternatively, the depressed reactions to some sensory stimuli could indicate decreased excitability, but there was no dose–response evident and the magnitude of these changes was small. In general, few of these behavioral effects displayed a clear relationship to dose. In males and females, 5MXC and 50MXC produced more significant effects than did 150MXC. A problem was the lower number of 150MXC rats (six males, seven females) which decreased the power of the statistical analyses. In addition, since mortality occurred at 150MXC, the remaining rats were survivors and may have simply been less sensitive to MXC. Nonetheless, most of the alterations were only marginal differences, the results were not dose-dependent, and there was a lack of concordance in other related endpoints. Differences in the ontogeny of sexually dimorphic behaviors could be suggestive of MXC-induced masculinization of females. This can be explained by the process found in normal CNS development, whereby testosterone is aromatized to estradiol (rev. in MacLusky and Naftolin,1981) to ‘‘set’’ a male pattern of behaviors. Providing supranormal amounts of estromimetics amplifies this process, as has been reported with perinatal exposure to zearalenone and estradiol to hamsters (Gray et al., 1985) and to chlordane, a cyclodiene pesticide shown to interact with steroid responses in vivo (Cassidy et al., 1994), but not with MXC (Gray et al., 1985). The lack of a clear profile or pattern of effects in the neurotoxicological evaluations is consistent with previous data, and could be due to insufficient doses and/or the type of tests. The latter may be the case, since the inclusion of

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

the neurological tests in this study design was to evaluate pesticide-induced changes in motor, sensory, autonomic, and cognitive function, but not specific sexually dimorphic behaviors (e.g., social and play behaviors) reported elsewhere to be affected by estrogenic compounds. The detection of estrogenic, or ‘‘endocrine-disrupting,’’ compounds, was the purpose of the reproductive endpoints, and indeed, numerous changes indicative of estrogenicity were observed. The monotonic reduction in antibody plaque-forming cells per spleen and per million spleen cells in males must be viewed in the context of a lack of effect in other related endpoints. If this effect were biologically significant, one might have expected to see alterations in the mixed lymphocyte response, the mitogen response, and or splenocyte surface marker expression (Luster et al., 1992). The lack of effect in the PFC response in females, as well as the lack of effect in other related endpoints, although compelling because they are dose-related and monotonic, may be anomalous. Estrogenic and androgenic hormones or xenobiotics reduce thymus weight by incompletely understood mechanisms (rev. in Silverstone, 1997). While the thymus weight reductions seen in both the female necropsies at 150MXC and in the pubertal male 150MXC group are consistent with these previous reports, they stand in contrast to the lack of consistent functional changes, as noted above. Further work will be necessary to define the scope and nature of these thymic effects, and determine their significance to the health of the animal. The greatest reproductive effects were seen in females: pnd46 ovary weight was reduced in all groups of dosed females, females at 150MXC were completely infertile, while females at 50MXC showed a pregnancy index similar to that seen for males at 150MXC (Tables 5 and 7). The number of live young produced by the 5MXC females (mean Å 11.5/litter) was not statistically different from controls (mean Å 13.8 implants/litter) in the second litter of this study, but decreases of this magnitude are statistically significant in the multilitter RACB design (see Morrissey et al., 1989), suggesting that with the greater statistical power available using multiple litters, reduced pup output would be detected even at the lowest dose of MXC tested. The subfertility and infertility of the 50 and 150MXC groups can be attributed to a lack of maturation of the ovarian–pituitary axis. This conclusion is supported by the temporal observations of the pnd42 females compared to the adults. The pnd42 ovaries were hypoplastic with delayed follicle maturation and complete anovulation. Adult ovaries were polycystic and lacked corpora lutea due to continued development of follicles but continued failure to ovulate. The process of ovulation is triggered by the feedback interactions of both the maturational signaling of the ovary and the pituitary. The pituitary produces FSH and lutenizing hormone (LH) to stimulate follicular growth. The maturing follicles produce estradiol and to a lesser extent progesterone which first inhibit and

ftoxal

153

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE

TABLE 7 Male Mating Trial Data in Adults (Group H) after Neonatal/Juvenile Methoxychlor Exposure Methoxychlor (mg/kg/day)

First mating trial: Males active (m) Mating Index (%) Females Pregnant/Cohabited Neonates PND 0 Live Dead PND 1 Live Dead PND 7 Live Dead Second mating trial Males active (m) Mating Index (%) Females pregnant/cohabited Live fetuses Dead fetuses Resorptions Implant sites Corpora lutea PreImplantation loss

0

5

50

150

13/15 93.3 21/30

15/15 86.7 22/30

14/16 75.0 20/30

2/15* 13.3* 3/30*

11.6 { 0.8a 0.20 { 0.08

12.4 { 0.7 0.18 { 0.11

12.6 { 0.7 0.35 { 0.17

13.7 { 0.7 0.0

11.5 { 0.8 0.09 { 0.06

12.3 { 0.8 0.09 { 0.30

12.6 { 0.7 0.05 { 0.05

13.7 { 0.7 0.0

11.7 { 0.6 0.05 { 0.05

12.8 { 0.5 0.0

12.4 { 0.6 0.0

13.7 { 0.7 0.0

13/15 86.7 18/29 13.2 { 0.1 0.06 { 0.06 0.6 { 0.2 13.8 { 0.5 17.1 { 0.7 2.5 { 0.6

14/15 93.3 20/30 14.2 { 0.9 0.0 0.5 { 0.2 14.6 { 1.0 18.4 { 0.6 3.0 { 0.9

14/16 87.5 22/32 12.1 { 0.9 0.0 0.5 { 0.1 12.6 { 0.9 18.4 { 0.6 5.4 { 1.2

4/15* 26.7* 6/30* 14.5 { 0.9 0.0 0.2 { 0.2 14.7 { 1.0 19.2 { 0.7* 2.3 { 1.3

Note. m, Males having at least one pregnant female. a Mean { SEM. * p õ 0.05 compared to controls.

then stimulate a surge of LH that triggers ovulation (rev. in Richards and Hedin, 1988). Exogenous chemicals could cause anovulatory cycles by directly targeting the ovary, the pituitary, or both the ovary and the pituitary. We believe that the anovulation due to MXC exposure in this study can be attributed to effects on the pituitary or hypothalamus. Levels of estradiol were the same between control and MXC treatment groups on proestrus and estrus, suggesting that ovarian follicles differentiated and functioned similarly regardless of MXC exposure. However, FSH levels were significantly suppressed in all treatment groups at estrus, including the 5MXC group in which ovarian weight and uterine weights were also reduced. If suppressed FSH levels were mediated by endogenous estradiol levels, then estradiol levels would necessarily be elevated in all MXC treatment groups, which was not observed. However, if the suppression of FSH was due to a direct ‘‘estrogenic’’ effect of MXC, then estradiol levels would be normal and FSH levels would be suppressed in all treatment groups, as they were. The suppression of estrus FSH levels may also explain delayed maturation of follicles in the pnd42 groups, since the estrus rise of FSH recruits follicles for the next cycle (Hirshfield, 1978). The significantly suppressed pro-

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

gesterone levels in the 50MXC and 150MXC groups compared to that seen in controls is attributed to the lack of ovulation and corpora lutea formation, since the 5MXC groups ovulated, formed corpora lutea, and had progesterone levels comparable to controls. Thus, the ovarian morphology and the comparisons of FSH, estradiol and progesterone levels are all consistent with a pituitary/hypothalamic defect. Studies using this exposure paradigm and specific hormonal manipulations (ovariectomy, hormonal challenge, etc.) would be required to confirm this tentative conclusion. The uterus was also directly affected: the reduced uterine weight seen in the pregnant rats starting at 5MXC (Table 6) is consistent with the earlier report for adult rats from Cummings and Gray (1987) at a substantially higher dose (¢200 mg/kg/day). This effect could not be explained by a difference in implant number, which was the same in the control and 5MXC groups. The uterine endometrial hyperplasia and squamous metaplasia is also consistent with a direct MXC effect on the uterus. Alternatively, uterine changes may reflect the altered estradiol:progesterone ratios in these animals, due to altered pituitary–ovarian feedback systems. Male fertility was affected only at 150MXC. Most male necropsy endpoints were significant at 150MXC, while testis

ftoxal

154

CHAPIN ET AL.

TABLE 8 Necropsy Data from Adult Males (Group H) after Neonatal/Juvenile Methoxychlor Exposure Methoxychlor (mg/kg/day) 0 Terminal weight (g) No. of rats Body weight Liver Absolute Relative (b) Kidney Absolute Relative Adrenals Absolute Relative Spleen Absolute Relative Thymus Absolute Relative Right testis Absolute Relative Right epididymis Absolute Relative Left cauda epididymis Absolute Relative Seminal vesicles Absolute Relative Prostate Absolute Relative Other data % Motile sperm Sperm per gram cauda (1105) Left testis weight Spermatid heads/gram testis (1105) Spermatid heads/total testis (1105)

509.0

5

15 { 9.9a

50

15 { 10.5

503.0

502.0

150

16 { 12.3

484.0

15 { 14.1

17.20 { 0.40 3.39 { 0.06

16.80 { 0.50 3.34 { 0.09

18.30 { 0.50 3.65 { 0.07

17.50 { 0.60 3.60 { 0.06

3.12 { 0.10 0.61 { 0.01

3.11 { 0.07 0.62 { 0.01

3.23 { 0.08 0.65 { 0.02

3.21 { 0.12 0.66 { 0.02

0.047 { 0.003 0.009 { 0.001

0.044 { 0.003 0.009 { 0.001

0.045 { 0.002 0.009 { 0.001

0.042 { 0.003 0.009 { 0.001

0.95 { 0.03 0.19 { 0.01

0.94 { 0.05 0.19 { 0.01

0.99 { 0.08 0.20 { 0.02

1.02 { 0.04 0.21 { 0.01

0.42 { 0.02 0.084 { 0.005

0.46 { 0.03 0.090 { 0.006

0.41 { 0.03 0.082 { 0.006

0.41 { 0.03 0.085 { 0.005

1.98 { 0.06 0.39 { 0.01

1.94 { 0.03 0.38 { 0.01

1.72 { 0.051* 0.34 { 0.01*

1.39 { 0.06* 0.29 { 0.01*

0.68 { 0.02 0.13 { 0.003

0.66 { 0.01 0.13 { 0.002

0.62 { 0.02* 0.12 { 0.004

0.53 { 0.01* 0.11 { 0.004*

0.236 { 0.008 0.047 { 0.002

0.232 { 0.008 0.046 { 0.002

0.213 { 0.006 0.043 { 0.001

0.184 { 0.009* 0.038 { 0.002*

2.41 { 0.13 0.48 { 0.03

2.09 { 0.07* 0.42 { 0.01

2.24 { 0.07 0.45 { 0.02

1.92 { 0.08* 0.40 { 0.02*

0.62 { 0.04 0.121 { 0.010

0.53 { 0.03 0.105 { 0.005

0.54 { 0.03 0.109 { 0.008

0.45 { 0.04* 0.094 { 0.009*

81.0 124.4 1.11 15.2 16.6

{ { { { {

1.8 70.0 0.04 1.1 1.2

80.0 116.8 1.09 13.8 15.1

{ { { { {

1.4 70.9 0.02 0.9 1.0

73.0 111.2 0.96 12.8 12.1

{ { { { {

2.9 68.2 0.03* 0.7 0.6*

70.0 82.6 0.76 13.5 10.7

{ { { { {

2.7* 8.7* 0.05* 1.2 1.1*

a

Mean { SEM. (Organ weight/corrected body weight) 1 100. * p õ 0.05 compared to controls. b

weight, epididymal weight, and spermatid heads/testis were also reduced at 50MXC. Most, but not all (e.g., epididymal weight and cauda epididymal sperm count) were affected at lower doses than reported previously (Gray et al., 1989). One might hypothesize that this is due, again, to the difference in exposure period. This difference in response due to critical window of exposure has been reported for numerous other reproductive toxicants, such as TCDD, nitrofen, and dibromochloropropane, inter alia (rev. in Gray, 1992). In the testis, the microscopic changes in testicular structure were minimal, and sperm output per gram of testis was

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

roughly equivalent across all groups (Table 8). However, there was a É35% reduction in epididymal sperm count and a 40% reduction in testicular spermatid numbers. This might be explained by a reduction in the total number of Sertoli cells, which might have been mediated by a reduction in prenatal levels of follicle stimulating hormone (rev. in Sharpe, 1993; Sharpe and Skakkebaek, 1993). Studies for determining Sertoli cell number in treated males are ongoing. Other male tissues showed no abnormal microscopic structure, and there were no indications of abnormal growth or differentiation. No Mullerian duct remnants were found

ftoxal

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE

FIG. 7. Methoxychlor effects on serum estradiol and progesterone levels in rats with estrus-like vaginal histology. Because higher doses of MXC inhibited cycling but resulted in cornified squamous vaginal epithelium characteristic of estrus, control and 5MXC rats in estrus were compared to 50 and 150MXC rats with estrus-like vaginal histology. Estradiol to progesterone ratios were compared by ANOVA after log transformation (log (Control[0]) Å 0.3 { 0.06 in n Å 7; log (5MXC) Å 0.13 { 0.09 n Å 7; log (50MXC) Å 1.27 { 0.09 n Å 17; log (150MXC) Å 1.19 { 0.07 n Å 15). Note that the 50 and 150MXC-treated rats failed to ovulate and thus had no ovarian source of progesterone. *Significantly different at p õ 0.05.

during necropsy, showing that these doses of MXC produced an effect different from those of DES (Newbold and McLachlan, 1988). No evidence of prostate dysplasia was observed. It is known that the first postnatal week is the time for steroid imprinting of the prostate in rats (Rajfer and Coffee, 1978; Chung and MacFadden, 1980). It has been

FIG. 8. Methoxychlor effects on serum FSH levels on proestrus or estrus. Rats treated with vehicle or 5 mg MXC/kg/day (n Å 6–8 per treatment group per time point) were necropsied during proestrus or estrus as determined by vaginal cytology and confirmed by vaginal histology. Serum FSH levels were compared according to stage by T test (JMP) after log transformation (Proestrus log (Control [0]) Å 0.37 { 0.035; log (5MXC) Å 0.39 { 0.047; Estrus log (Control [0]) Å 0.79 { 0.03; log (5MXC) Å 0.57 { 0.05). *Significantly different at p õ 0.05.

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

155

FIG. 9. Methoxychlor effects on serum FSH levels in rats with estruslike vaginal histology. Because higher doses of MXC inhibited cycling but resulted in cornified squamous vaginal epithelium characteristic of estrus, control and 5MXC rats in estrus were compared to 50 and 150MXC rats with estrus-like vaginal histology. Serum FSH levels were compared by ANOVA after log transformation (log (Control [0]) Å 0.79 { 0.03 in n Å 6; log (5MXC) Å 0.57 { 0.05 n Å 7; log (50MXC) Å 0.33 { 0.04 n Å 16). *Significantly different at p õ 0.05.

shown that direct dosing of neonates with estrogens can modify this imprinting, altering androgen receptor levels (Prins and Birch, 1995) and subsequent growth patterns (Kincl et al., 1963; Prins et al., 1996, inter alia). The lack of effect observed in the present study might be due to (1) evaluating the animals at too young an age, (2) insufficient estrogenic signal transmitted to the neonatal prostates to produce a detectable effect with these methods since the dams were dosed during the first week, and MXC and metabolites are relatively weak estrogen receptor agonists, or (3) inadequate tissue sampling. Based on recent reports of minute estrogenic stimulation altering prostate responses later in life (vom Saal et al., 1997), another study is currently underway to evaluate specifically prostatic structure and immunohistochemistry using the present exposure paradigm. There were several endpoints for which effects were observed even at 5 mg MXC/kg/day: pnd46 ovary weights, empty adult uterus weight and metaplasia, age at vaginal opening, and FSH during estrus. While the vaginal opening change is probably not adverse, it is more difficult to consider that the reduced organ weights and hormonal stimuli do not represent the beginnings of adverse effects, particularly in populations of animals. Additional analysis is required to determine just how big a change will correlate with altered function over multiple studies. Our attempts to apply benchmark dose methodology to these data to estimate a NOAEL were frustrated by the shapes of the dose–response curves, which evidenced significant nonlinearity using the control, low, and middle dose data (R. W. Morris, unpublished).

ftoxal

156

CHAPIN ET AL.

These data lower, but do not define, the NOAEL for MXC. The IRIS database reports that the lowest NOAEL is a rabbit teratology study with a NOAEL level of 5 mg/kg/day; all other reproductive data have NOAELs higher than this value. The data from the present study confirm that exposures starting before birth can have profound and long-lasting effects on the structure and function of the rodent reproductive system. ACKNOWLEDGMENTS The authors gratefully acknowledge the input of Drs. Jim Rowe and Sue MacMasters, and W. C. Williams and M. M. Riddle, of the U.S. EPA. Analysis for methoxychlor and metabolites were performed by Cedra Corporation, Austin, TX, under Contract NO1-ES-25332. We also gratefully acknowledge the help of of Mr. Al Caviness and Ms. Kathy Feola at NIEHS, Msrrs. Michael Vesselica and Charlie Sparracino, and Dr. Bob Handy at Research Triangle Institute for chemistry support under NIEHS Contract NO1-ES15307. Warm thanks are also due to Ms. Beth Gaul and Drs. Larry Johnson and Bob Maronpot for discussions during the course of this work and to Drs. Earl Gray, Mike Shelby, and Susan Makris for their review of the manuscript.

REFERENCES Anscombe, F. J. (1948) The transformation of Poisson, binomial and negative binomial data. Biometrika 35, 246–254. Appel, R. J., and Eroschenko, V. P. (1992). Passage of methoxychlor in milk and reproductive organs of nursing female mice. 1. Light and scanning electron microscopic observations. Reprod. Toxicol. 6, 223–231. Archer, J. (1973). Tests for emotionality in rats and mice: A review. Anim. Behav. 21, 205–235. Armitage, P. (1971). Statistical Methods in Medical Research, pp. 362– 365. Blackwell Scientific, Great Britain. Benjamins, J. A., and McKhann, G. M. (1981). Development, regeneration, and aging of the brain. In Basic Neurochemistry (G. J. Siegel, R. W. Albers, B. W. Agranoff, and R. Katzmann, Eds.), (3rd ed.) pp. 445–469. Little Brown, Boston. Bulger, W. H., Muccitelli, R. M., and Kupfer, D. (1978). Studies on the in vivo and in vitro estrogenic activities of methoxychlor and its metabolites. Role of hepatic mono-oxygenase in methoxychlor activation. Biochem. Pharm. 27, 2417–2423. Bulger, W. H., Feil, V. J., and Kupfer, D. (1985). Role of hepatic monooxygenases in generating estrogenic metabolites from methoxychlor and from its identified contaminants. Mol. Pharm. 27, 115–124. Cassidy, R. A., Vorhees, C. V., Minnema, D. J., and Hastings, L. (1994). The effects of chlordane exposure during pre- and postnatal periods at environmentally relevant levels on sex steroid-mediated behaviors and functions in the rat. Toxicol. Appl. Pharm. 126, 326–337. Chapin, R. E., Harris, M. W., Shelby, R. J., Smialowicz, V. C., Moser, S., Padilla, R. C., MacPhail, A. C., Lockhart, and Mauney, M. C. (1996). The effects of perinatal/juvenile pesticide exposure on adult CNS, Immune, and reproductive function in rats. Fundam. Appl. Toxicol. 30(Suppl.), 52. Chung, L. W. K., and MacFadden, D. K. (1980). Sex steroids imprinting and prostatic growth. Invest. Urol. 17, 337–342. Clemens, L., Gladue, B., and Coniglio, L. (1978). Prenatal endogenous androgenic influences on masculine sexual behavior and genital morphology in male and female rats. Horm. Behav. 10, 40–53. Creason, J. (1989). Data evaluation and statistical analysis of functional observational battery data using a linear models approach. J. Am. Coll. Toxicol. 8, 157–170.

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

Cummings, A. M. (1993). Replacement of estrogen by methoxychlor in the artificially-induced decidual cell response in the rat. Life Sciences 52, 347–352. Cummings, A. M., and Gray, L. E. J. (1987). Methoxychlor affects the decidual cell response of the uterus but not other progestational parameters in female rats. Toxicol. Appl. Pharmacol. 90, 330–336. Dixon, W., and Massey, F. (1951). Introduction to Statistical Analysis, pp. 145–147. McGraw Hill, New York. Dostal, L. A., Weaver, R. P., and Schwetz, B. A. (1990). Excretion of high concentrations of cimetidine and ranitidine into rat milk and their effects on milk composition and mammary gland nucleic acid content. Toxicol. Appl. Pharmacol. 102, 430–442. Dunnett, C. W. (1955). A multiple comparisons procedure for comparing several treatments with control. J. Am. Stat. Assoc. 50, 1096–1121. Anon. (1994). Environ. Health Perspect. 102(8), 628–629. Goldman, J. M., Cooper, R. L., Rehnberg, G. L., Hein, J. F., McElroy, W. K., and Gray, L. E, Jr. (1986). Effects of low subchronic doses of methoxychlor on the rat hypothalamic pituitary reproductive axis. Toxicol. Appl. Pharmacol. 86, 474–483. Gray, L. E., Jr. (1992). Chemical-induced alterations of sexual differentiation: a review of effects in humans and rodents. In Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection (T. Colborn and C. Clement, Eds.). Princeton Scientific, Princeton, NJ. Gray, L. E., Jr., Ferrell, J. M., and Ostby, J. S. (1985). Alteration of behavioral sex differentiation by exposure to estrogenic compounds during a critical neonatal period: Effects of zearalenone, methoxychlor, and estradiol in hamsters. Toxicol. Appl. Pharmacol. 80, 126–136. Gray, L. E., Jr., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., Goldman, J., Slott, V., and Laskey, J. (1989). A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundam. Appl. Toxicol. 12, 92–108. Greco, T. L., Duello, T. M., and Gorski, J. (1993). Estrogen receptors, estradiol, and diethylstilbestrol in early development: The mouse as a model for the study of estrogen receptors and estrogen sensitivity in embryonic development of male and female reproductive tracts. Endocr. Rev. 14, 59–71. Harris, M. W., Chapin, R. E., Lockhart, A. C., and Jokinen, M. P. (1992). Assessment of a short-term reproductive and developmental toxicity screen. Fundam. Appl. Toxicol. 19, 186–196. Harris, M. W., Chapin, R. E., Haskins, E. A., Allen, J. D., Collins, B. J., Davis, B. J., Lockhart, A. C., and Mauney, M. (1996). The effects of perinatal/juvenile pesticide exposure on adult reproductive function. I. Methoxychlor. Fundam. Appl. Toxicol. 30(Suppl.), 144. Harris, S. J., Cecil, H. C., and Bitman, J. (1974). Effect of several dietary levels of technical methoxychlor on reproduction in rats. J. Agr. Food Chem. 22, 969–973. Hirschfield, A. (1978). The role of FSH in the selection of large ovarian follicles in the rat. Biol. Reprod. 19, 606–611. Hodge, H. C., Maynard, E. A., Thomas, J. F., Blanchet, H. J. J., Wilt, W. G. J., and Mason, K. E. (1950). Short-term oral toxicity tests of methoxychlor (2,2 di-(p-methoxy phenyl)-1,1,1-trichlorethane) in rats and dogs. J. Pharmacol. Exp. Ther. 99, 140–148. Jones, I. C. (1955). Role of the adrenal cortex in reproduction. Br. Med. Bull. 11, 156–160. Joyce, K. L., Hess, R. A., and Cooke, P. S. (1996). Neonatal estrogen treatment effects on Sertoli cell proliferation and testis development in the rat. J. Androl. (Suppl.1), 5, 24. Kapoor, I. P., Metcalf, R. L., Nystrom, R. F., and Sangha, G. K. (1970). Comparative metabolism of methoxychlor, methiochlor, and DDT in mouse, insects, and in a model ecosystem. J. Agr. Food Chem. 18, 1145– 1152.

ftoxal

ADULT EFFECTS AFTER JUVENILE MXC EXPOSURE Kincl, F. A., Folch-Pi, A., and Herrera Lasso, L. (1963). Effect of estradiol benzoate treatment in the newborn male rat. Endocrinology 72, 966– 968. Korenbrot, C. C., Huhtaniemi, I. T., and Weiner, R. I. (1977). Preputial separation as an external sign of pubertal development in the male rat. Biol. Reprod. 17, 298–303. Luster, M. I., Munson, A. E., Thomas, P. T., Holsapple, M. P., Fenters, J. D., White, K. L., Lauer, L. D., Germolec, D. R., Rosenthal, G. J., and Dean, J. H. (1988). Development of a testing battery to assess chemicalinduced immunotoxicity: National Toxicology Program’s guidelines for immunotoxicity evaluation in mice. Fundam. Appl. Toxicol. 10, 2–19. Luster, M. I., Portier, C., Pait, D. G., White, K. L., Jr., Gennings, C., Munson, A. E., and Rosenthal, G. J. (1992). Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam. Appl. Toxicol. 18, 200–210. MacLusky, N. J., and Naftolin, F. (1981). Sexual differentiation of the central nervous system. Science 211, 1294–1303. McDaniel, K. L., and Moser, V. C. (1993). Utility of a neurobehavioral screening battery for differentiating the effects of two pyrethroids, permethrin and cypermethrin. Neurotoxicol. Teratol. 15, 71–83. Morrissey, R. E., Lamb, J. C., IV, Morris, R. W., Chapin, R. E., Gulati, D. K., and Heindel, J. J. (1989). Results and evaluations of 48 continuous breeding reproduction studies conducted in mice. Fundam. Appl. Toxicol. 13, 747–777. Moser, V. C., McCormick, J. P., Creason, J. P., and MacPhail, R. C. (1988). Comparison of chlordimeform and carbaryl using a functional observational battery. Fundam. Appl. Toxicol. 11, 189–206. National Academy of Sciences (1993). Pesticides in the Diets of Infants and Children. National Academy Press, Washington, DC. Neter, J., Wasserman, W., and Kutner, M. H. (1985). Applied Linear Statistical Models, 2nd. ed. Richard D. Irwin, Inc., Homewood, IL. Newbold, R. R., and McLachlan, J. A. (1988). Neoplastic and non-neoplastic lesions in male reproductive organs following perinatal exposure to hormones and related substances. In Toxicity of Hormones in Prenatal Life (T. Mori and H. Nagasawa, Eds.), pp. 89–109. CRC Press, Boca Raton. Prins, G. S., Birch, L., Ye, S. H., and Ray, V. (1996). Neonatal estrogen exposure leads to prostate lobe-specific dysplasia and adenomas in the aging rat. J. Androl. (Suppl.1) 118, 52. Prins, G. S., and Birch, L. (1995). The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen. Endocrinology 136, 1303–1314. Public Health Service (1996). Policy on Humane Care and Use of Laboratory Animals. Office for Protection from Research Risks, NIH, Rockville, MD. Rajfer, J., and Coffey, D. S. (1978). Sex steroid imprinting of the immature prostate: Long-term effects. Invest. Urol. 16, 186–190. Reiter L. W. (1983). Chemical exposures and animal activity: Utility of the figure-eight maze. In Developments in the Science and Practice of

AID

FAAT 2381

/

6k23$$$281

11-10-97 09:54:57

157

Toxicology (A. W. Hayes, R. C. Schnell, and T. S. Miya, Eds.), pp.73– 84. Elsevier Science, New York. Research Triangle Institute (1995). Priority Data Needs for Methoxychlor. U.S. Dept. of Health and Human Services. Richards, J. S., and Hedin, L. (1988). Molecular aspects of hormone action in ovarian follicular development, ovulation and luteiniztion. Annu. Rev. Physiol. 50, 441–463. Robb, G. W., Amann, R. P., and Killian, G. J. (1978). Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J. Reprod. Fertil. 54, 103–107. SAS User’s Guide (1990). SAS Institute, Cary, NC. Sharpe, R. M. (1993). Declining sperm counts in men—Is there an endocrine cause? J. Endocrinol. 136, 357–360. Sharpe, R. M., and Skakkebaek, N. E. (1993). Are œstrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet 341, 1392–1395. Silverstone, A. E. (1997). T-cell development. In Comprehensive Toxicology (D. A. Lawrence, I. G. Sipes, C. A. McQueen, and A. J. Gandolfi, Eds.), Vol. 5, pp. 39–56. Elsevier Science, London. Smialowicz, R. J., Rogers, R. R., Rowe, D. G., Riddle, M. M., and Luebke, R. W. (1987). The effects of nickel on immune function in the rat. Toxicology 44, 271–281. Smialowicz, R. J., Andrews, J. E., Riddle, M. M., Rogers, R. R., Luebke, R. W., and Copeland, C. B. (1989). Evaluation of the immunotoxicity of low level PCB exposure in the rat. Toxicology 56, 197–211. Smialowicz, R. J., Riddle, M. M., Luebke, R. W., Copeland, C. B., Andrews, D., Rogers, R. R., Gray, L. E., and Laskey, J. W. (1991). Immunotoxicity of 2-methoxyethanol following oral administration in Fischer 344 rats. Toxicol. Appl. Pharmacol. 109, 494–506. Smialowicz, R. J., Riddle, M. M., Williams, W. C., and Diliberto, J. J. (1994). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on humoral immunity and lymphocyte subpopulations: Differences between mice and rats. Toxicol Appl. Pharmacol. 124, 248–256. Tilson, H. A., and Moser, V. C. (1992). Comparison of screening approaches. Neurotoxicology 13, 1–14. Tullner, W. W. (1961). Uterotrophic action of the insecticide methoxychlor. Science 133, 647–648. Tullner, W. W., and Edgcomb, J. H. (1962). Cystic tubular nephropathy and decrease in testicular weight in rats following oral methoxychlor treatment. J. Pharmacol. Exp. Therap. 138, 126–130. U.S. Environmental Protection Agency. (1991). Revised neurotoxicity test guidlines. Pesticide Assessment Guidelines, Subdiv. F, Hazard Evaluation: Human and Domestic Animals, Addendum 10, publication PB-91154617. National Technical Information Service, Springfield, VA. vom Saal, F., Timms, B. G., Montano, M. M., Palanzo, P., Thayer, K. A., Nagel, S. C., Dhar, M. D., Ganjam, V. K., Parmigiani, S., and Welshons, W. V. (1997). Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses. Proc. Natl. Acad. Sci. USA 94, 2056–2061. Welch, R. M., Levin, W., and Conney, A. H. (1969). Estrogenic action of DDT and its analogs. Toxicol. Appl. Pharmacol. 14, 358–367.

ftoxal