Perinatal hexachlorobenzene toxicity in the mink

ENVIRONMENTAL

RESEARCH

Perinatal

31, 116- 124 (1983)

Hexachlorobenzene

Toxicity

in the Mink

GLENN F. RUSH, JACQUELINE H. SMITH, KEIZO MAITA, MICHAEL BLEAVINS, RICHARD J. AULERICH, ROBERT K. RINGER, JERRY

B.

AND

HOOK

Departments of Pharmacology, Toxicology, and Animal Science, Center for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824 Received

May

6, 1982

Adult female standard dark mink were exposed to hexachlorobenzene (HCB) at concentrations of 0, 1, and 5 ppm in the feed and bred with mates on the same treatments. Female offspring were allowed to mature to 16- 17 weeks and killed. At 16- 17 weeks of age, HCB had no effect on body weights or liver weights. Hepatic cytochrome P-450 and ethoxyresorufin-O-deethylase were significantly increased 2.0- and l.Sfold, respectively, in the 5-ppm treatment group. Electron microscopy failed to reveal proliferation of the smooth endoplasmic reticulum. No hepatic damage was observed. No changes in in vitro renal function, measured as accumulation of para-aminohippurate and tetraethylammonium by renal cortical slices, were detected in any treatment group. Histological examination of renal slices did not reveal any alterations in morphology. Fat was the predominate site of HCB disposition; samples from the 5-ppm treatment group contained 626.10 * 12.01 ng HCB/g tissue. Whereas perinatal HCB administration has profound effects on the survival of offspring born to exposed mink, only induction of hepatic mixed-function oxidases was observed in the surviving kits without any observable frank hepatotoxicity.

INTRODUCTION

Hexachlorobenzene (HCB) has become widespread in the environment through its use in agriculture as a fungicide and in industry in the production of chlorinated solvents. Residues of HCB have been reported in a variety of wild animals (Koss and Manz, 1976), dairy products (Smyth, 1972), and human fat and milk (Bakken and Seep, 1976; Brady and Siyali, 1972). Once in the environment, HCB is degraded very slowly and bioaccumulates. The most prevalent toxic manifestations of HCB in mammals are hepatomegaly, porphyria, and immunosuppression (Courtney, 1979; Loose et al., 1977). Predatory animals that are near the top of their food chain can accumulate and biomagnify HCB (Courtney, 1979; Koss and Manz, 1976) possibly resulting in toxicity. The mink is an upper level, native North American carnivore of economical importance. This species is especially susceptible to halogenated hydrocarbon toxicity, particularly embryotoxicity (Aulerich et al., 1973; Aulerich and Ringer, 1977). In the mid- 196Os, mink ranchers experienced a decline in the reproduction of animals (Hartsough, 1965) which was eventually traced to the high PCB content of Great Lakes fish that was part of the commercial diet. Mink fed polychlorinated biphenyls (PCBs, Aroclor 1242) experimentally at 5 ppm in the diet have complete reproductive failure (Bleavins et al., 1980). This adverse effect of PCBs on reproduction is apparently not due to alterations in the male reproductive system since 116 0013-9351/83 $3.00 Copyright @ 1983 by Academic Press, Inc. AU rights of reproduction in any form reserved.

HEXACHLOROBENZENE

TOXICITY

IN

MINK

117

nontreated females mated with treated males had normal litters (Aulerich and Ringer, 1977). Recently, similar effects on reproduction have been observed with HCB, as mortality of kits to weaning from mink fed 5 ppm in the diet was 77.4% (Bleavins, unpublished observation). The purpose of this study was to determine the effects of HCB perinatally administered in surviving kits from mink fed 1 and 5 ppm in the diet. In addition, tissue distribution of HCB was also determined. METHODS Animals. Adult standard dark mink (Must&a vision) were used in these experiments. Experimental diets were prepared by incorporating the appropriate amount of HCB (Pfaltz and Bauer, Stamford, Conn.) into 500 g of commercial mink cereal (XK-40 Grower, XK Mink Foods, Thiensville, Wise.) and adding this to the basal wet diet.’ Animals were assigned to groups fed diets containing either 0 (control), I, or 5 ppm HCB in a chronic feeding trial initiated on January 20, 1981. Food and water were provided ad fibitum. Adult mink were bred in March and whelped their litters in late April and early May. The mink kits were weaned onto nonsupplemented basal diet on June 15, 1981. Young mink were therefore exposed to HCB via three routes: (1) in utero throughout gestation, (2) their dam’s milk during lactation, and (3) from HCB-contaminated feed they may have consumed directly, prior to weaning. Eighteen female mink born to dams described above (six from each of three treatment groups) were weighed and killed at 16- 17 weeks of age. Blood, fat, muscle, kidney, and brain samples were taken for HCB analysis. Livers and kidneys were removed and immediately placed in ice-cold 0.9% saline. Hepatic mixed-function oxidases. Microsomes from mink liver were prepared according to Dent et al. (1980). In a typical reaction, microsomes (0.25-0.5 mgml) were suspended in 1 ml of 66 mM Tris-HCl buffer (pH 7.8) containing 4.5 PM glucose &phosphate, 0.3 PM NADP, 0.3 PM NADH, 0.1 PM NADPH, 163 PM MgCl,, and 1 unit of glucose-dphosphate dehydrogenase. After 3 min preincubation at 37”C, the reaction was initiated by the addition of substrate. Enzymatic activities measured were ethoxyresorufin-0-deethylase (Johnson et al., 1979), ethoxycoumarin-0-deethylase (Aitio, 1978), and benzphetamine-N-demethylase (Prough and Ziegler, 1977). Cytochrome P-450 and cytochrome b5 were measured according to the method of Omura and Sato (1964). NADPH cytochrome c reductase was measured by the method of Pederson et al. (1973). Microsomal protein was determined by the method of Lowry et al (1951). In vitro renal function. Thin renal cortical slices were prepared free hand. Organic ion accumulation was determined by incubating SO- 150 mg of slices in 4 ml of medium composed of 96.7 mM NaCl, 7.4 mM sodium phosphate buffer (pH 7.4), 40 mM KCl, and 0.74 mM CaCl, (Cross and Taggart, 1950). The medium was supplemented with 10 mM lactate (Sigma Chemical Co., St. Louis, MO.) for one half of the slice incubations and also contained 7.4 X 10m5 M p-aminohippurate 1 The basal diet consisted of 20% whole chicken, 16.7% commercial mink cereal, 12.5% ocean fish scrap, 6.7% beef liver, 6.7% beef lungs, 3.3% beef tripe, 3.3% beef trimmings, 2.9% cooked eggs, 1.1% powdered milk, and 26.8% added water.

118

RUSH

ET

AL.

(PAH) and 1 x 10m5 M [lJ4C]tetraethylammonium (TEA), sp act 2.00 mCi/mmol (New England Nuclear, Boston, Mass.). After incubation for 90 min at 25°C under 100% 0, in a Dubnoff metabolic shaker, slices were removed from the medium, blotted, and weighed. Slices were homogenized in 6 ml of 5% trichloroacetic acid and brought up to volume of 10 ml with distilled water. A 2-ml aliquot of the incubation medium was treated similarly. After centrifugation the supernatant was assayed for PAH by the method of Smith et al. (1945) an for TEA by liquid scintillation spectrometry. PAH or TEA accumulation in kidney slices was expressed as the slice to medium or S/M ratio. Glucose synthesis by renal cortical slices was determined by a method similar to that of Roobol and Alleyne (1974). The incubation medium consisted of a Krebs-bicarbonate medium containing 10 mM pyruvate as the substrate for gluconeogenesis. After gassing the medium with 02-CO.,,, 50- 100 mg of slices was placed in 5 ml of the medium in a 50-ml Erlenmeyer flask, flushed with 02-COz, stoppered tightly, and incubated at 37°C for 1 hr in a Dubnoff metabolic shaker. After incubation, the slices were removed from the medium, blotted, and weighed. The medium glucose content was determined using glucose oxidase and peroxidase as described in Sigma Technical Bulletin No. 510 (l-78) (Sigma Chemical Co., St. Louis, MO.). Tissue analysis of HCB. Concentrations of HCB in liver, kidney, fat, muscle, brain, and blood were determined using the following method. Samples were weighed and ground with sufficient anhydrous sodium sulfate to render them completely dry and pulverized. Powdered tissues were extracted three times with 40-ml portions of hexane. Pooled extracts were reduced in volume to l-2 ml and placed on a Florisil(60to loo-mesh) column (500 x 200 mm). HCB was eluted with 200 ml of hexane which was evaporated to a l-ml volume, then brought up to the desired volume. Quantitation was by gas-liquid chromatography on a Varian Model 2100 gas chromatograph (Varian, Palo Alto, Calif.) with a Sc3H electron capture detector using a 1.7-m x 2-mm-id. column packed with 3% OV-225. Carrier gas (N& flow and column temperature were 30 ml/min and 165”C, respectively. Concentrations were expressed as nanograms of HCB per gram of tissue wet weight. Histological Examination. Several thin slices of liver and kidney were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and processed for routine light microscopic observation including fat staining with Oil red 0. For electron microscopy, tissue slices were diced into l-mm blocks, dipped in 4% glutaraldehyde/O. 1 M cacodylate buffer fixative, and placed into fresh fixative for 3 hr. Tissue blocks were then washed in Zetterquists’ buffer twice, placed in 1% 0~0, solution for 1.5 hr, and embedded into Epon-Alaldite plastic after dehydration through graded solutions of alcohol and propylene oxide. Thin sections were then cut on an LKB-III ultratome, stained with uranyl acetate/lead citrate, and examined on a Zeiss electron microscope (9S2) operated at 60 kV. Statistical analysis. Data were analyzed statistically by an analysis of variance, completely random design. Treatment differences were detected by the least significance difference test (Steel and Tot-tie, 1960). The 0.05 level of probability was used as the criterion of significance.

HEXACHLOROBENZENE

TOXICITY

IN

119

MINK

RESULTS HCB had profound effects on the survival of kits to weaning as mortality increased from 8.2% in control to 44.10 and 77.4% in the l- and 5-ppm treatment group, respectively. At 17 weeks of age, surviving kits from mink fed 0, 1, and 5 ppm HCB had no alterations in body weight, or kidney or liver weights (Table I). Microsomes from control livers contained 0.080 + 0.010 nmol cytochrome P-450 (P-450)/mg protein and kits from the 5-ppm treatment group had elevated hepatic P-450 concentrations (0.160 +_ 0.014 nmol/mg protein). Cytochrome b5 was not altered in any treatment groups (Fig. 1). Correlating with an increased hepatic P-450, hepatic ethoxyresorufin-0-deethylase in the 5-ppm group increased 1.5fold over controls. No changes were observed in ethoxycoumarin-0-deethylase, benzphetamine-ZV-deethylase, and NADPH cytochrome-c reductase (Table 2). No alterations in in vitro renal function were observed as determined by accumulation of PAH or production of glucose by renal cortical slices in any treatment group. TEA accumulation was slightly, but significantly decreased in kidneys from the 5-ppm treatment group (Table 3). HCB was primarily distributed in fat in all treatment groups with detectable amounts in brain, liver, kidney, and muscle (Table 4). HCB could not be detected in blood from any treatment group. Light microscopy of hepatic cells revealed numerous small cytoplasmic vacuoles which stained red with Oil red 0, however, no treatment differences were observed (data not shown). Electron microscopy of liver showed that almost the entire cell cytoplasm is occupied by elipsoidal vesicular profiles of various sizes (from 250 mprn to 7 pm in diameter) (Fig. 2, A and B). These vesicles were largely empty except for occasional deposition of fine electron-dense granules and were not membrane enclosed. There were no alterations in Golgi apparatus, mitochondria, or other major cytoplasmic organelles including endoplasmic reticulum due to HCB (Fig. 2). No alterations were observed in either light or electron micrographs from kidney (data not shown). DISCUSSION Aulerich et al. (1973) first reported a decrease in the reproductive success of female mink fed a basal diet that contained whole fish from the Great Lakes region. It was later determined that PCBs in the fish were responsible for mink reproductive failure (Aulerich and Ringer, 1977). Administration of 5 ppm Aroclor

TABLE

1

EFFECTOPPRENATAL HEXACHLOROBENZENEADMINISTFCATION ON BODYANDORGANWEIGHTS Treatment (pm HCB) 0 1 5 Note.

N = 6.

Body wt (kid 0.73 k 0.02 0.75 k 0.09 0.67 f 0.05

Liver Body

wt x loo wt

2.64 2 0.12 2.83 k 0.07 2.69 k 0.08

Kidney Body

wt wt

x loo

0.49 -c 0.03 0.54 f 0.01 0.49 -+ 0.03

120

RUSH ET AL.

0 fm

1ppm

5ppm

FIG. 1. The effect of perinatal HCB exposure in the diet on hepatic cytochrome P-450 (open bars) and cytochrome b5 (hatched bars). Asterisk indicates significantly different from control (P < 0.05).

1242 to mink in the diet results in total reproductive failure (Bleavins et al., 1980). Administration of equivalent doses of HCB does not interfere with reproductive success; however, survival of kits to weaning is markedly affected. In the present study, surviving kits from dams fed 1 and 5 ppm HCB displayed no indication of toxicosis. Ultrastructural changes have been observed in rat liver after 5 ppm HCB in the diet as proliferation of the smooth endoplasmic reticulum (SER) and abnormal mitochondrial morphology (Mollenhaver et al., 1975). While proliferation of hepatic SER was not observed in any treatment group, hepatic P-450 and ethoxyresorufin-0-deethylase were induced in kits from dams fed 5 ppm HCB. Phenobarbital and 3-methylcholanthrene induced mink hepatic P-450 but there appears to be a very limited substrate specificity as few enzymatic activities increase @hull, personal communication). Ethoxyresorufin-0-deethylase, which was increased in the present studies, is a 3-methylcholanthrene-inducible enzyme whereas benzphetamine-Ndemethylase, which was not changed, is phenobarbital inducible. In the rat, HCB increases enzymatic activity consistent with a phenobarbital-type of induction but results in a shift in spectral characteristics of TABLE EFFECT

2

OF PRENATAL HEXACHLOROBENZENE HEPATIC MIXED-FUNCTION

TreatmenP (mm HCB)

NADPH cytochrome c reductaseb

Ethoxyresorufin0-deethylase

Control 1 5

56.31 ? 12.00 70.61 & 5.41 70.77 2 4.20

1.64 t 0.35 2.35 f 0.20 2.52 f oso*

ADMINISTRATION OXIDASES

Ethoxycoumarin0-deethylasti 0.94 2 0.11 1.18 f 0.14 0.88 k 0.09

ON

BenzphetamineN-demethylasec 4.17 + 1.90 6.08 -c 2.01 6.17 t 2.11

Note. N = 6. a Pregnant mink received hexachlorobenzene in the diet at concentrations of 0, 1, and 5 ppm. b nmol cytochrome c reducedlminlmg protein. c nmol/min/mg protein. * Significantly different from control (P < 0.05).

HEXACHLOROBENZENE

EFFECT

TOXICITY

121

MINK

TABLE 3 OF PRENATAL HEXACHLOROBENZENE ADMINISTRATION ON RENAL GLUCONEOGENESIS ACCUMULATION OF p-AMINOHIPPURATE AND TETRAETHYLAMMONIUM BY MINK RENAL CORTICALSLICE~ -Lactate

Treatment” @pm

IN

HCW

Control 1 5

GluconeogenesisC 4.34 f 0.27 3.78 r 0.18 3.97 * 0.41

PAH

SIM

2.5 + 0.1 2.6 k 0.1 2.7 t 0.2

+Lactate TEA

S/M

13.1 + 1.0 13.2 5 1.0 12.7 + 0.8

PAH

S/M

13.6 2 2.1 16.8 2 1.0 15.6 t 1.0

a Values represent mean f SEM of at least three animals. * Hexachlorobenzene administered to pregnant mice in diet at concentration C mg glucose/g tissue/hr. * Significantly different from control (P < 0.05).

P-450 to a lower wavelength

AND

TEA

S/M

20.2 -c 0.2 18.9 5 1.0 17.0 2 0.3*

of 1 and 5 ppm.

similar to that seen after 3-methylcholanthrene (Stonard and Nenov, 1974; Courtney, 1979) and has been termed a “mixed” type inducer. In the mink, HCB appears to behave predominately as a 3-methylcholanthrene-type mixed-function oxidase inducer. Pregnant mice administered pentachloronitrobenzene (PCNB) contaminated with 10% HCB demonstrated a high incidence of renal agenesis in offspring whereas no alteration was observed after technical-grade PCNB (Courtney et al., 1976). It was concluded that the renal teratogenic effects were due to HCB. In addition, female Rhesus monkeys exposed to HCB showed renal histological alterations (Iatropoulos et al., 1976). In contrast, in the present study, perinatal HCB exposure had no effect on in vitro renal function or histology. The reason for the high mortality among nursing mink remains unclear. The apparent lack of toxicity among surviving mink may reflect their relatively low body burden of HCB, as kits from dams fed 5 ppm had only 0.6 ppm in the body fat and very low levels in other tissues. It is likely that kits received the majority of their HCB body burden during lactation, since in the mouse, toxic doses of HCB can be readily transmitted via the milk as young, nontreated mice, cross-fostered with lactating HCB-treated mothers had a high rate of mortality (Courtney, 1979). Alternatively, these surviving mink had a marked T-cell suppression (Bleavins, unpublished observation). Immunosuppression from HCB exposure may have contributed to the high incidence of mortality prior to weaning. In conclusion, HCB, while causing a high degree of mortality of young mink prior to weaning, resulted in no significant hepatic or renal damage in surviving mink. Increased xenobiotic metabolism in these animals may alter their response to toxic agents in the environment known to be metabolically activated. Altematively, induction of hepatic mixed-function oxidases in these mink may result in certain endocrine-related disorders through altered metabolism of steroid hormones.

35.71 _’ 12.81 94.59 f 17.30 626.10 + 12.01

0 1 5

ND ND 36.31 * 3.52

Braid

Live9

ND 4.18 f 0.98 14.61 + 5.01

Kidney

FOLLOWINGPRENATALADMINISTRATION

4

ND ND 36.71 r 13.48

TABLE OFHEXACHLOROBENZENE

Note. N = 6, ND = not detected. o Pregnant mink were treated with 1 and 5 ppm HCB in the diet. * ng hexachlorobenzene/g tissue.

FaP

DISTRIBUTION

TreatmenP @pm HCB)

TISSUE

Blood* ND ND ND

MusclZ ND 1.42 -c 0.50 8.45 e 0.75

F

rl

z is

FIG. 2. Electron in the diet.

microscopy

of liver

from

mink

perinarally

exposed

to 0 ppm HCB

(A) or 5 ppm (B)

124

RUSH

ET AL.

ACKNOWLEDGMENTS The authors wish to thank V. G. Adler and T. Hopkins for their expert technical assistance and Diane Hummel for manuscript preparation. This work is Michigan Agriculture Experimental Station No. 10411.

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