Animal toxicology of chlorinated dibenzo-p-dioxins

Chemosphere,Vol.12,No.4/5,pp Printed in Great Britain

453-462,1983

OO45-6535/1983/O50453-iO$O3.OO/o ©1983 Pergamon Press Ltd.

ANIMAL TOXICOLOGYOF CHLORINATEDDIBENZO-p-DIOXINS

H. Poiger and Ch. Schlatter I n s t i t u t e of Toxicology, Swiss Federal Institute of Technology and University of Zurich, CH-8603 Schwerzenbach, Switzerland

When discussing animal t o x i c i t y of PCDDs, i t must be kept in mind that the bulk of data available apply to the 2,3,7,8-isomer (TCDD), the most toxic out of the 75 congeners of PCDDs. I t is of particular importance that there are substantial differences between animal species with respect to their s e n s i t i v i t y to PCDDs. Furthermore, the types of toxic responses to these compounds d i f f e r between species and include acute, chronic, genotoxic and immunotoxic effects. Some of these investigations are reviewed with special attention given to genotoxic effects. Own recent investigations exhibited that metabolites of 2,3,7,8-TCDD are less toxic than the parent compound and also that the rate of metabolism and b i l i a r y excretion of metabolites in the dog is stimulated by TCDD i t s e l f . ACUTE AND SUBCHRONIC EFFECTS Out of the 75 isomers of chlorinated dibenzo-p-dioxins and 125 isomers of chlorinated d i benzofurans only a small number has been tested by classical toxicological testing procedures. A series of experiments has been conducted by McConnell et al. [ I ] to compare the acute t o x i c i t y levels of some of the PCDDcongeners {Table I ) . The data show marked differences in the doses required to produce l e t h a l i t y . The most toxic compound is 2,3,7,8-TCDD (TCDD) which, therefore {and for other reasons), has i n i t i a t e d a considerable number of toxicological and other studies. Deletion of a chlorine at a lateral position resulted in a marked loss in t o x i c i t y ; addition of a chlorine atom at the ortho position reduced t o x i c i t y to a lesser extent. The data also indicate that the two rodent species used are unequally sensitive towards the compounds. But each of the toxic chlorinated dioxins w i l l induce all the toxic responses produced by TCDD, i f administered in a s u f f i c i e n t dose. The data on halogenated dibenzofuranes and biphenyl congeners are more limited but also suggest that those congeners isosteric with TCDD produce the same toxic syndrome. Table I: Single oral LD50_30 Values of Chlorinated Dioxins {pg/kg) Chlorination

Guineapig

2, 8 300 000 2,3,7 29 444 2,3,7,8 2 1,2,3,7,8 3.1 ],2,4,7,8 1 125 I, 2,3,4,7,8 72.5 1,2,3,6,7,8 70-100 1,2,3,7,8,9 60-100 1,2,3,4,6,7,8 600 I-N02-2,3,7,8 47.5 { 2,3,7,8-TCDF)* 5-10 *Data from Moor et al. [2]

453

Mouse -3 000 283.7 337.5 5 000 825 I 250 1 440 -2 000 6 000

454

A lot more data are available on the suceptibility of species towards the most toxic isomer, TCDD, being compiled in Table 2. For instance, in the guinea pig the single oral dose LD50 is 0.6-2 pg/kg whereas the Golden Syrian hamster is much less sensitive with an LD50 being about 3-4 orders of magnitude greater. Table 2: Acute Toxicity of 2,3,7,8-TCDD in Different Species Species, strain Guinea piq, Hartley m, Rat, Sherman m, f, Rat, Sprague Dawleym, f, weanling m, Chicken, Monkey, Macacamulatta, Rabbit, New Zealand albino, Mouse, C57B]/Sch strain m, Mouse, C57B1/6fh (J67) m, Dog, Beagle, Golden Syrian hamster m,f Golden Syrian hamster m

Single-dose LD50 (~g/kg) 0.612.0 22 45 60 25 25 25-50 ca 70 115 275 114 284 ca 200-300 3000 1157 5051

R o u t e Reference p.o. p.o. p.o. p.o. p.o. p.o. p.o. p.o. p.o. dermal p.o. p.o. p.o.

r3,]] [31 r4] F57 F6]

r3] r7] [I] F3]

p.o.

[el

i.p. p.o.

r9]

However, the acute t o x i c i t y of TCDD and related compounds cannot be viewed in the classical sense of an LD50, because the onset of the toxic response is very slow and, after application of a lethal dose, an extended time of several weeks lapses until death occurs. The marked species differences raise doubts on a common mechanism for the toxic action. In most cases the t o x i c i t y in laboratory animals is characterized by a marked, sometimes biphasic, weight loss {25-30 % of bw), a phenomenonwhich has been observed not only with TCDD but also with other congeners as well as PCDFs. Thvmic atrophy appears to be a constant finding in a l l species F9, 10, 11]. A significant TCDD-induced hepatic damagehas been observed in the rat, rabbit, mouse and dog, but not in the monkey, guinea pig and hamster. In chicken, charact e r i s t i c lesions included ascites or edema, signs which were also observed after treatment with hexa-CDD F121. In monkeys, effects on the bone marrow and e p i t h e l i a l tissue are more prominent. But in most cases the degree of tissue destruction is insufficient to define the cause of death. The ultimate target organ is unknown, making i t d i f f i c u l t to know which t i s sue to focus on. The pathological appearancegives the impression of a starvation-like syndrome despite other species specific syndromes. In more chronic exposure, bile duct hyperplasia is found to varying degrees in all animal species, especially pronounced in nonhuman primates FS]. In a subchronic t o x i c i t y study in rats Kociba et al.F13] administered 1, 0.1, 0.01 and 0.001 pg TCDD/kg/d, 5 days per week for 13 weeks. Some mortality, decreased body weight and feed consumption, thymic atrophy, icterus, hepatic lesions, increased urinary excretion of porphyrins and ~-Ala were observed at the highest dose level. At 0.1 pg/kg there was a doserelated decrease in the toxic response, whereas no toxic effects could be observed at 0.01 and 0.001 pg/kg. Clinical chemistry changes generally reflect the underlying histopathologic alterations in various organs. For example, increased levels of GOT [14] GPT, ~r-GT, alkaline phosphatase and b i l i r u b i n [13, 551 were reported in rats. In guinea pigs TCDD fnduced a marked hyperlipidemia, resulting from mobilization of adipose tissue f a t t y acids [15]. Chronic treatment with TCDD caused porphyria and increased excretion of porphyrins in mice [16, 17] and female rats F18, 191. I t has been observed that an iron deficient diet protects against porphyria due to TCDD and that susceptibility to porphyria requires AHH-responsiveness F16]. ENZYME INDUCTION TCDD and analogues have been found to be potent inducers of a battery of enzymes involved in xenobiotic metabolism F20, 21, 22]. Especially the inductive effect on AHH-activity in l i v e r and other tissues of several species has been noted by many investigators. For various halogenated dibenzo-p-dioxins the structure-activity relationship for induction

455

of hepatic AHH in chicken embryos have been examined by Poland et al. F25]. A similar induction pattern has also been measured in v i t r o in rat hepatoma cell cultures F23G. The potency of these compounds to induce AHH-activity, in certain species, was found to correlate with their toxic potency F25], but interestingly the rabbit and the guinea pig, very sensitive species, seem to be less responsible to enzyme induction F24]. A number of studies have demonstrated the existence of a cytosolic protein, which reversibly binds these compounds with a high a f f i n i t y F26]. This protein appears to be the receptor for enzyme induction. Poland and G1over F39] proposed that the t o x i c i t y of TCDD and congeners is mediated through this receptor. We were interested to know, whether l i v e r metabolism of TCDD, which has been shown to occur at a slow rate in rats F27, 28] and at considerable rates in hamsters and dogs [29, 30] is influenced by inducing agents. Studies of Beatty et al. [4] have demonstrated a decreased s e n s i t i v i t y of rats towards TCDD after pretreatment with MFO-stimulating agents, which might be a consequence of an increased metabolism and subsequent excretion of the dioxin. In our laboratory most recently some experiments were undertaken to investigate the influence of a pretreatment with phenobarbital or TCDD i t s e l f on the rate of TCDD metabolism and b i l i a r y excretion of metabolites in the dog.

5v IfW I0 ~g unlabelled TCDD/kg ....~ | 9 days earlier .o.~'" I \ .I ~30~ o~ u | 200~ 1/ O 14

.s'" Figure I: Cumulative Biliary Excretion of ~.r no pretreatment TCDD-Metabolites in D~gs After /" ~,".... 4 Single Oral Doses of -H-TCDD i (30 ng/kg bw). ~ C I/kg/d 38

62

86

ii0 t (h)

Figure 1 shows the cumulative b i l i a r y excretion profiles of 3H-activity from 3 experiments, carried out subsequently in a male boxer dog. The excreted material (biotransformation products) was referred to the absorbed amount of the TCDD-dose, since intestinal absorption varied considerably. Pretreatment with phenobarbital (20 mg/kg/d for 10 days) had no effect, whereas a single dose of 10 pg/kg of unlabelled TCDD (9 days prior to the labelled TCDD) resulted in a marked increase in b i l i a r y metabolite excretion. Whether this increased elimination has r e a l l y an important effect on the t o x i c i t y of TCDD in the dog cannot be concluded from these experiments. Generally, in view of the striking differences in susceptibility among species towards TCDD i t remains questionable whether different biotransformation rates could give an adequate explanation at a l l . On the other hand we could demonstrate that TCDD metabolites, isolated from the bile of TCDD-dosed dogs do not belong to the same class of toxicants than the parent compound. Dose$of up to 100 times the LD50 of TCDD-metabolites did not reveal a toxic response in male guinea pigs (Table 3). The deaths observed in the control and highest dose group were found to be caused by material coextracted from the bile (one animal in the control group died after receiving an amount of bile extract corresponding to the highest dose group *) as well as from a handling error. No gross pathological or histologic changes could be observed in the principal TCDDtarget organs. Table 3: Mortality and Body Weight Gain in Male Guinea Pigs Following a Single Oral Treatment with TCDD-Metabolites Dose pg/kg bw

No. of death/ no. of treated

0 0 0 0.6 6.0 30.0 60.0 Weber et al. [31]

0/5 0/5 I/4" 0/5 0/5 0/5 2/7

Mean body weight gain (g15 weeks) 313 242 177 307 302 315 210

456

TERATOLOGY AND REPRODUCTI~ EFFECTS Again, most of the data, concerning this area, are available from the tetrachloro-isomer. Fortunately, results from animal studies are much clearer than data from man where the question of teratogenic and reproductive effects is highly controversial. In animals, teratogenic effects are rather uniform for mice, where c l e f t palate and kidney abnormalities predominate. These are common malformations induced by a wide range of unspecific factors in this species. A summary of studies is listed in Table 4, which indicate that the no-adverse effect level for teratogenic response in the mouse is at about O.l pg/kg/day, which is a factor being at least 10 times lower than the toxic concentration for adult mice. Interestingly, co-administration ofp-naphtoflavone, which is another ligand of the Ah-receptor increased the frequency of clefi~ palate formation when compared with the number increased by TCDD alone F36]. Table 4: Teratology Studies with TCDD in Mice and Rats Species, strain

Teratogenic effects

Mouse CD-I DBA/2J C57B1/6J NMRI CB57BI/6

Cleft palate, kidney malformations

CF-I NMRI C57BL Rat Spr.Dawley CD Wistar

Dose pg/kg

I, 3

Cleft palate 3/9 Cleft palate, kidney 1, 3 malformations Cleft palate I, 3 Cleft palate 16 Cleft palate 25 Intestinal hemorrh. 0.125-8 Kidney malformations 0.5 Visceral anomalies 0.25-16 (no alive fetuses/surviv. pups at doses >1 pg/kg)

Time TCDD given, d

Route

Ref.

6-15

s.c.

[32]

6-15,9-13 10, 10-13

p.o. p.o.

[33] [34]

p.o. i.p. i.p. p.o. s.c. p.o.

[351 [36]

6-15 10-13 11-13 6-15 6-15 6-15

[37] F32] F38]

Some further evidence that TCDD-evoked c l e f t palate formation segregates with the Ah-locus is given by studies of Poland and Glover F39]. Data displayed on Table 5 show that among strains with a low a f f i n i t y receptor, TCDD produced a 0-3% incidence of c l e f t palates. Four of the f i v e strains tested with a high a f f i n i t y receptor developed a 54-95 % incidence in response to TCDD. Table 5: Cleft Palate Formation in Ten Inbred Strains of Mice Strain

Numberof litters

C57BL/6J A/J BALB/cByJ SEC/IReJ CBA/J

13 7 9 4 12

12 16 11 6 5

52/96 37/51 26/40 19/20 0/61

54 73 65 95 0

DBA/2J RF/J AKR/J SWR/J

16 9 8 7

7 18 I 3

2/96 1/34 0/38 0/37

2 3 0 0

3

0/29

O

129/J 6 Poland & Glover F39]

Deador resorbed Cleft palate/ fetuses live fetuses

Percentage c l e f t palate

No teratogenic response was observed with 2,7-diCDD, 1,2,3,4-tetraCDD, a mixture (40:60) of 2,7-diCDD/2,3,7-triCDD as well as with the octa-congener in CD-I mice F40]. In rats, TCDD appears to be more fetotoxic than teratogenic, producing intestinal hemorrhage and edema, kidney abnormalities and internal hemorrhages. The no-adverse effect level for the rat embryo lies below 0.125 ~g/kg/day (Table 4).

457

In a three generation reproduction study with Sprague Dawley rats [413, where TCDD was feed in the diet, providing dose levels of 0.1, O.Ol and 0.001 pg of TCDD/kg/day, the breeding performance was disrupted at the O.l pg dose level. L i t t e r size at birth, gestation survival, neonatal survival and qrowth were decreased. F e r t i l i t y was decreased in FI and F2 but not in FO generations. No effects on the reproductive capacity was observed at the lowest dose level. No dose related fetal abnormalites were seen in this study. Adverse reproductive effects have been reported also in nonhuman primates (Table 6): Rhesus monkeys, fed diets with 500 ppt TCDD had decreased serum estradiol and progesterone levels. After breeding to control males, three of 8 females conceived after which 2 aborted and I had a normal birth. After feeding a diet with 50 ppt, serum estradiol and progesterone levels were normal. Mating of 8 females resulted in 6 pregnancies, after which there were 4 abortions and 2 normal births. A no-effect dose in monkeys has not been determined [42]. The teratogenic potential of some other congeners has also been studied in the rat [3]: At a lO0 ug/kg dose level of hexaCDD (p.o. on days 6 through 15 of gestation) a decreased fetal body weight and an increased incidence of certain soft tissue and skeletal anomalies was observed, but also signs of t o x i c i t y in dams. 2,7-diCDD and octaCDD both neither caused teratogenicity nor embryotoxicity at 100 mg/kg/day. Unfortunately, up to our knowledge, no teratoloqy studies have been performed with the highly sensitive guinea pig and the relat i v e l y insensitive hamster. I t would be important to know, whether the wide differences in sensitivity, seen in the adult species somehowparallels with the sensitivity of the embryos. Table 6: Reproductive Performance of Rhesus Monkeys Fed TCDD in the Diet for Seven Months Prior to Conception and During Pregnancy

Total impregnated Absorptions/resorptions Stillborn Normal births Allen e t ' a l . F42]

50 ppt

500 ppt

6/8 4/8 0/8 2/8

3/8 2/8 0/8 I/8

Is TCDD a teratoqen ? Yes, a strong one, when the absolute quantities are used as c r i t e r i o n , but only a weak one, when the ratio of teratogenic and toxic dose is used for classification. MUTAGENICITY Only four dioxin isomers have been evaluated so far for mutagenicity, 2,7-diCDD, TCDD, octaCDD and the unsubstituted dibenzo-p-dioxin. In in v i t r o tests with Salmonella typhimurium strains (Table 7) negative results were reported for the unsubstituted dibenzo-p-dioxin [43]. TCDD was f i r s t l y evaluated for mutagenicity by Hussain et al. F45], who found increased mutation frequencies in TA 1532, a strain that detects frameshift mutations. The results of a study by Seiler [46] confirmed this finding in the same strain. A doubtful response was reported in strains TA 1531 and TA 1532; (a questionable response was also found for octaCDD in TA 1532 and TA 1534 strains). Later on, however, Seiler was not able to confirm his own e a r l i e r positive results. Unfortunately, these later findings have never been published. Other reports F44, 47, 48] could not demonstrate any mutagenic a c t i v i t y of TCDD in a l l the strains as indicated in Table 7. Studies in other test systems than Salmonella are summarized in Table 8. TCDD had a very weak prophage inducing effect in E.coli K 39 cells [45]. Jackson [49] reported that TCDDproduced a marked i n h i b i t i o n of mitoses in dividing endosperm cells of the African bloody l i l y as well as chromosome abnormalities. Mitotic gene conversion and reverse mutation in the D 7 strain of S. cerevisiae with $I0 fraction as well as in vivo with the intrasanguinated host mediated assay were reported by Bronzetti et al. F5O]. When rats were treated o r a l l y with 10 pg of TCDD/kg for 5 consecutive days (or with 2,7-diCDD and dibenzo-p-dioxin, respectively), no chromosomal alterations were observed in bone marrow cells [511, but treatment for 13 weeks twice weekly (2 or 4 ~g TCDD/kg) caused a weak increase in chromosome breaks in the rat bone marrow F521. In a variety of mammalian cell cultures, Beatty et al. F53] could not find any electron microscopic evidence of an altered cell morphology. Knutson and Poland F54] also could not detect toxic effects of TCDD in 23 cultured cell types, which were derived from tissues and/or species susceptible for TCDDt o x i c i t y in vivo. So one can conclude that TCDD is at least not a classical mutagen.

458

Table 7: Mutaqenicity of Dioxin-Compounds in Salmonella Compound

Strains with pos. mutagenic response

Dibenzo-pdioxin TCDD TCDD TCDD TCDD

TA 1532 TA 1532 (TA 1531, TA 1534 ?)

Strains with negative mutagenic response TA 1535, TA lO0, TA 1537 TA 1538 TA ]530 G 46, TA ]530

TA 1535, TA 1538 TA 98, TA 100, TA TA 1535, TA 1532, TA 1538, TA ]950, TA 1978, G 46 TA 1535, TA 1537, TA 98, TA 100 (TA 1532,TA ]534 ?) G 46, TA 1530, TA

TCDD octaCDD

1530 TA 1537 TA 1975

Ref. F431 F45] F461 [44] F471

TA 1538

[48]

1531

F46]

Table 8: Mutagenicity of TCDD in Various Test Systems Test System Prophage induction in E. coli K-39 Cytologic effects in African bloody l i l y Mitotic gene conversion and reverse mutation in S. cerevisiae D 7 Analysis of rat bone marrow, single appl. of TCDD (or dibenzo-p-dioxin, 2,7-diCDD) Analysis of rat bone marrow, 13-week administration Morphology of animal and human cells in culture Morphology, v i a b i l i t y and growth rate in 23 animal and human cell cultures

Result +-i n h i b . o f mitoses + -chromos, breaks

Ref. [451 F49] F50] F511 F52]

--

F531

--

F54]

CARCINOGENICITY TCDD: The results of two key carcinogenicity studies, conducted in Sprague Dawley [55] and Os--sB-orne Mendel rats F561 are summarized in Table 9. Both studies indicate an oncogenic response at the highest (O.l pg TCDD/kg/day via the diet and 0.5 pg/kg/week by gavage twice weekly, respectively) and at the medium dose level (O.Ol pg/kg/day and 0.05 pg/kg/week, respectively), though the increased incidence of thyroid adenoma at the medium dose level in the Osborne Mendel rats remained questionable. The l i v e r was the primary target tissue for oncogenesis in both strains. No increase in preneoplastic lesions was reported at the low dose levels (0.001 pg/kg/day and 0.01 pg/kg/week, respectively). Interestingly, Kociba et al. F551 observed also a decreased occurence of numerous age-related lesions usually encountered in the strain used, including tumors of the p i t u i t a r y , uterus, mammary gland, pancreas and adrenal gland at the high dose level. Carcinogenicity data in mice (Table 10) correlate quite well. At a dose level of O.l pg/kg/ day Toth et al. F57] observed an increased incidence of hepatocellular tumors in Swiss mice, though this strain had a r e l a t i v e l y high incidence of spontaneous l i v e r tumors. No increase was observed at the high dose level (1 pg/kg/day), possibly a result of the decreased l i f e span of the animals, nor at the low (0.001 pg/kg/day) dose level. Other data, originating from the NCI study F58] show an increase of the incidence of several tumor types at the high dose level (2 pg/kg/week in females and 0.5 pg/kg/week in males, by gavage twice weekly). No oncogenic response occurred at dose levels of 0.01-0.2 pg/kg/week.

459

Table 9: Carcinogenicity of TCDD in Rats Dose pg/kg/d

Strain Sprague Dawley m,f

0.1

Osborne Mendel m,f

0.01 0.001 0.071 0.007 0.0014

Response

Ref.

Hepatocellular carcinoma, [55] squamous carcinoma of lung, hardpalate, nasal turbinates Decreased: Tumors of p i t u i t a r y , uterus, mamm.gland, pancreas, adrenal gland. Hepatocellular nodules No increase in tumors Hepatocellular carcinoma, [56] thyroid adenoma, tissue and subcutaneous fibroma Questionableincrease in thyroid adenoma No increase in tumors

Table 10: Carcinogenicity of TCDD in Mice Species

Dose pg/kg/d

Swiss/H/Riop m

1.0 0.I

B6C3F1 f

0.001 0.29

m

0.071

f m f m

0.029 0.007 0.006 0.0015

Response

Ref.

Tumor incidence not increased [57] (but decreased lifespan) Increased incidence of hepatoc e l l u l a r tumors Tumor incidence not increased Increased incidence of hepato [56] c e l l u l a r tumors, thyroid and adrenal adenoma Increased incidence of hepatoc e l l u l a r tumors Tumor incidence not increased

Other dioxins: To date, reports on possible carcinogenicity are available for the unsubstituted dibenzo-p-dioxin, 2,7-diCDD and for a mixture of 1,2,3,6,7,8- and 1,2,3,7,8,9-hexaCDD (31% and 67 % of total hexaCDD, respectively). From a study, feeding a diet with 5000 or 10 000 ppm dibenzo-p-dioxin i t was concluded that this compound was not carcinogenic for Osborne Mendel rats and B6C3FI mice F58]. 2,7-diCDD, incorporated into the diet at both the same levels was concluded not to be carcinogenic in Osborne Mendel rats or female B6C3FI mice but may be carcinogenic in male B6C3FI mice F59]. The hexaCDD-mixturewas tested at dose levels of 1.25, 2.5 and 5 pg/kg/week (twice weekly by gavage) in rats and mice. The incidence of hepatocellular carcinoma was significantly increased at the mid-and high-dose levels in female but not in male rats. Both male and female mice showed a significant increase in hepatocellular adenoma at the highest dose level F60]. In a dermal painting study in Swiss Webster mice the incidence of the various neoplasms observed was not s i g n i f i c a n t l y affected [61]. Tumor I n i t i a t i o n and Promotion: Several attempts were made to decide whether PCDDs act as i n i t i a t i n g agents or indirect through promotion of already i n i t i a t e d cells. Someof the experimental data are summarized in Table I I : In v i t r o covalent binding of TCDD to r a t - l i v e r DNA has been found insufficient to explain oncogenesis of TCDD by this mechanism [62]. In numerous skin painting studies in mice

460

conflicting results have been reported. Studies of DiGiovanny F631 indicated TCDDto be only a weak i n i t i a t o r when followed by T-phorbol acetate (TPA) as promoting agent and application concurrently with DMBAfollowed by TPA revealed only a very slight increase of the number of papillomas when compared with the i n i t i a t i n g a b i l i t y of DMBA alone. Pretreatment with TCDD inhibited tumor i n i t i a t i o n by DMBA, BaP and MCA F64, 65]. The mechanism of this effect might be due to an increase in epidermal AHH-and other enzyme a c t i v i t i e s , since the inhibitory effect of TCDD was highIv dependent on the time of treatment r e l a t i v e to the i n i t i a t o r . L i t t l e or no effect was observed when TCDD was given 5 minutes before or I day after i n i t i a t i o n . Interestingly i t has been found that the quantity of 3H-DMBA bound to DNA and RNA of the epidermis was decreased in animals treated with TCDD, whereas binding of BaP was increased which suggests a difference in the metabolic activation or detoxification of DMBAversus BaP. In the test system used by Kuori et al. F661 TCDD, given 2 days prior to MCA did not affect the MCA carcinogenic index in genetically "responsive" (to AHH induction) C57BL6 as well as "nonresponsive" DBA/2 mice, even at doses of 100 Hq/kg which k i l l e d 30-70 % of the animals in each group. TCDD given simultaneously with MCA increased the index in DBA/2 mice. TCDD did not promote mouse skin tumors following DMBA i n i t i a t i o n and did not produce papillomas by i t s e l f in studies by Berry et al. F67]. In a recent study by the NCI F68] TCDDwas found not to be carcinogenic in male mice, but caused fibrosarcomas in the integumentary system in female mice. In a 2-stage system using rats F6g] TCDD acted as a potent promoter of l i v e r tumors, induced by DEN and partial hepatectomy, which stands in contrast to the results of Berry et al. r67] in the meuse skin tumorigenesis system. Few data are available on the promotion of skin tumors by other dioxins F70]: Following i n i t i a t i o n with DMBA, promotion with octaCDD had no effect, but treatment with diCDD and the unsubstituted dibenzo-p-dioxin yielded apparent skin tumors in male Swiss Webster mice. Table 11: TCDD: Tumor I n i t i a t i o n , Promotion and Cocarcinogenesis Species

Experimental design

Rat, Sprague Dawley m, f

In vivo covalent binding of TCDD to l i v e r DNA and rRNA

Mouse, CD-I,

2 pg TCDDon skin as i n i t i ator, TPA as promoter 2 pg TCDD + DMBAon skin as i n i t i a t o r , TPA as promoter Pretreatment with 1 ~g TCDD topically, DMBAas i n i t i a t o r TPA as promoter dito, BaP as i n i t i a t o r

f

Mouse, Sencar, f

Mouse, CD-I,

f

Pretreatment with I pg TCDD i . p . , i n i t i a t i o n with DMBA, promotion with TPA dito, BaP as i n i t i a t o r dito, MCA as i n i t i a t o r

Mouse, C57BL/6 and DBA/2, m,f Mouse, CD-I,

f

Mouse, Swiss Webster m, f Rat, Charles River f

l or 100 pg TCDD/kg i.p. or s.c. 2 days before MCA or with MCA (s.c.) DMBA on skin as i n i t i a t o r TCDD as promoter (0.1 pg twice weekly on skin) DMBAon skin as i n i t i a t o r TCDD as promoter (f:O.O05 ~g, m:O.O01 pg thrice wkly) P a r t i a l l y hepatectomized animals, 10 mg/kg DEN p.o. promotion with 0.14 or 1.4 ~g TCDD s.c, biweekly

Results Oncogenesis through covalent binding of TCDDto DNA is unlikely Very weak tumor i n i t i a t i o n by TCDD Slight increase of tumors compared to DMBAalone TCDD strongly inhibits DMBA tumor i n i t i a t i o n TCDD inhibits BaP tumor initiation TCDD inhibits DMBAtumor initiation TCDD inhibits BaP tumor initiation TCDD inhibits MCA tumor initiation TCDD not carcinogenic but may be a cocarcinoqen

Ref. F62] F63]

F64]

r65]

r66]

No promotion of skin tumors by TCDD

[67]

f : TCDDcarcinogenic m: TCDDnot carcinogenic

[68]

TCDD is a potent promoter for hepatocarcinoqenesis

F69]

461

IMMUNUTOXICITY The observation that intoxication with PCDDs causes thymic atrophy and depression of other l~mphatic tissues in a wide range of species has initiated a series of immunotoxicity studies, including functional and morphologic assessment of humoral and cell-mediated immunity. However, there are a number of contradictory findings and definite conclusions cannot be drawn. Thymosin as well as growth hormone had no effects on thymus atrophy due to TCDD, nor were zinc levels depressed [71]; adrenal and p i t u i t a r y gland did not appear to be involved in causing thymic involution F727. Suppression of cell-mediated immunity and increased susceptib i l i t y to bacterial infections have been described F73, 74, 75]. Immunosuppressive effects were most pronounced after animals were exposed to TCDD in utero, or in young adult animals after chronic treatment. I t has frequently been assumed that TCDD does not markedly affect humoral response FI, 75, 76], but mot recent results of Vecchi et al. [77] showed that single doses of TCDDmarkedly suppressed primary humoral antibody production in 6-8 week old mice. CONCLUSION Although much data have been accumulated so far, the mode of action of TCDD and other PCDDs is s t i l l unknown. This question remains to be a challenge for toxicologists. Of more practical importance is the lack of knowledge of the toxicology of other isomers. In this area we need the help of synthetic chemists who w i l l provide us with sufficient quantities of these compounds to perform toxicological studies. These data then w i l l allow us to properly assess the health immplications of the dioxins occurring in our environment. REFERENCES: FI]

E.E. McConnell, J.A. Moore, J.K. Haseman, and M.W. Harris, Toxic. appl. Pharmacol., 4_44, 335 (1978). F27 J.A. Moore, E.E. McConnell, D.W. Dalgard, and M.W. Harris, Ann. N.Y. Acad. Sci., 320, 151 (1979). F3] B.A. Schwetz, J.M. Norris, G.L. Sparschu, V.K. Rowe, P.J. Gehring, J.L. Emerson, and C.G. Gerbiq, Environ. Hlth Perspec., 5, 87 (19731. F4] P.W. Beatty, W.K. Vaughn, and R.A. Neal, Toxic. appl. Pharmacol., 45, 513 (19781. F57 E.E. McConnell, J.A. Moore, and D.W: Dalgard, Toxic. appl. Pharmaco--T., 43, 175 (1978). F6] J.G. Vos, J.A. Moore, and J.G. Zinkl, Toxic. appl. Pharmacol., 29, 229-F1974). F77 J.R. Olson, M.A. Holscher, and R.A. Neal, Toxic. appl. Pharmaco~, 55, 67 (19801. F81 J.W. Henck, M.A. New, R.J. Kociba, and K.S. Rao, Toxic. appl. Pharm~., 59, 405 (19811. [9] B.N. Gupta, J.G. Vos, J.A. Moore, J.G. Zinkl, and B.C. Bullock, Environ.~Ith Perspec., 5, 125 (1973). F107 I~.H. Norback and J.R. Allen, Environ. Hlth Perspec., _5, 233 (19731. [117 R.D. Kimbrough, Crit. Rev. Toxicol., 2, 445 (19741. [12] J.R. Allen and J.J. Lalich, Proc. Soc_ Exp. Biol. Med., 109, 48 (19621. [13] R.J. Kociba, P.A. Keeler, C.N. Park, and P.J. Gehring, Toxic. appl. Pharmacol., 3__55,553 (1976). F14] N.P. Buu-Hoi, Naturwissenschaften, 59, 174 (19721. [15] L.L. Swift, T.A. Gasiewicz, G.D. Du~, P.D. Soul~, and R.A. Neal, Toxic. appl. Pharmacol., 59, 489 (1981). FI6] K.G. Jones, ]~7.M. Cole, and G.D. Sweeney, Toxic. appl. Pharmacol., 61, 74 (1981). [17] J.A. Goldstein, P. Hickman, H. Bergman, and J.G. Vos, Res. Commun. ~l~em. Pathol. Pharmacol., 6, 919 (1973). [18] J.A. GoldsteTn, P. Hick~n, and H. Bergman, Fed. Proc. 35, 708 (1976). [19] L. Cantoni, M. Salmona, and M. Rizzardini, Toxic. appl.-llharmacol., 5_77, 156 (1981). F201 K.T. Kitchin and J.S. Woods, Molec. Pharmacol., 14, 890 (19781. F21] A. Poland and E. Glover, Molec. Pharmacol., 9, 7 ~ (1973). F22] B.V. Madhukar and F. Matsumura, Toxic. appl.-Pharmacol., 61, 109 (19811. F237 J.A. Bradlaw, L.H. Garthoff, and N.E. Hurley, Fd Cosmet. ~ x i c o l . , 18, 627 (19801. F241 G.E.R. Hook, J.K. Hasema,, and G.W. Lucier, Chemico-Biol. I n t e r a c t i ~ s , 10, 199 (19751. F251 A. Poland, W.F. Greenlee, and A.S. Kende, Ann. N.Y. Acad. Sci., 320, 214~19791. F267 A. Poland, E. Glover, and A.S. Kende, J. Biol. Chem., 251, 4936 ~ 7 6 ) . F277 H. Poiger and Ch. Schlatter, Nature, 281, 706 (19791. - F28] J.C. Ramsey, J.G. Hefner, R.J. Karbows-~, W.H. Braun, and P.J. Gehring, Toxic. appl. Pharmac., 65, 180 (1982). F29] J.R. Olson-,--T.A. Gasiewicz, and R.A. Neal, Toxic. appl. Pharmacol., 5_66, 78 (1980).

462

F307 H. Poiger, H.-R. Buser, H. Weber, U. Zweifel, and Ch. Schlatter, Experientia, 3__88,484 11982). F317 H. Weber, H. Poiger, and Ch. Schlatter, Toxicol. Lett., 14, 117 (1982). F327 K.D. Courtney and J.A. Moore, Toxic. appl. Pharmacol., ~-7396 {197I). F337 D. Neubert and H. Dillman, Arch. Pharmacol., 272, 243 (~72). F347 J.A. Moore, B.N. Gupta, J.G. Zinkl, and J.G. ~ , Environ. Hlth Perspec., 5, 81 (1973). F357 F.A. Smith, B.A. Schwetz, and K.D. Nitschke, Toxic. appl. Pharmacol., 38, 517 (1976). F367 E.M. Hassoun and L. Dencker, Toxicol. Lett., 12, ]91 {1982). F377 G.L. Sparschu, F.L. Dunn, and V.K. Rowe, Fd C~met. Toxicol., 9, 405 (1971). F387 K.S. Khera and J.A. Ruddick, in: Chlorodioxins-Origin and Fate? Advances in Chemistry Series, No. 120 (ed. E.H. Blair): Washington, D.C., (1973). F397 A. Poland and E. Glover, Molec. Pharmacol., 17, 86 (]980). F407 K.D. Courtney, Bull. Environ. Contam. ToxicoT? 16, 674 (1976). F41] F.J. Murray, F.A. Smith, K.D. Nitschke, C.G. Hum-~-ston, R.J. Kociba, and B.A. Schwetz, Toxic. appl. Pharmacol., 50, 241 (]979). F42] J.R. Allen, D.A. Barsotti~-L.K. Lambrecht, and J.P. Van Miller, Ann. N.Y. Acad. Sci., 320, 419 (1979). F437 B. Commoner, EPA-Report No. EPA-600/I-76-022 (1976). F447 D.W. Nebert, S.S. Thorgeirsson and J.S. Felton, in: In vitro Metabolic Activation in Mutagenesis Testing, (F.J. de Serres, J.R. Fouts, J.R. Bend, and R.M. Philpot eds.): Elsevier/North-Holland, Amsterdam, p. 105 (1976). F457 S. Hussain, L. Ehrenberg, G. L~froth, and T. Gejvall, Ambio, i , 32 (1972). F467 J.P. Seiler, Experientia, 29, 622 (]973). F477 P. Gilbert, G. Saint-Ruf, F__ Poncelet, and M. Mercier, Arch. Environ. Contam. Toxicol., 9, 533 (1980). F48] I.E. Geiger and R.A. Neal, Toxic. appl. Pharmacol., 59, 125 (1981). F49] W.T. Jackson, J. Cell Sci., 10, 15 (1972). F507 G. Bronzetti, I. Lee, E. Z e i ~ r , H. Malling, and K. Suzuki, Mutat. Res., 74, 206 (1980). F517 S. Green and F.S. Moreland, Toxic. appl. Pharmacol., 33, 161 (1975). F527 S. Green, F.S. Moreland, and C. Shen, FDA By-Lines, 6~--292 (1977). F537 P.W. Beatty, K.J. Lembach, M.A. Holscher, and R.A. Neal, Toxic. appl. Pharmacol., 3_I], 309 (1975). F547 J.C. Knutson and A. Poland, Toxic. appl. Pharmacol., 54, 377 (1980). F557 R.J. Kociba, D.G. Keyes, J.E. Beyer, R.M. Carreon, C.~Wade, D.A. Dittenber, R.P. Kalnins, L.E. Frauson, C.N. Park, S.D. Barnard, R.A. Hummel, and C.G. Humiston, Toxic. appl. Pharmacol., 46, 279 C]978). F567 NTIS/PB82-163445, ~ v t . Reports Announcements & Index, 11 (1980). F577 K. Toth, S. Somfai-Relle, J. Sugar, and J. Bence, Nature-~, 278, 548 (]979). F58] National Cancer Institute, Carcinog. Tech. Rep. Set., I 2 2 ~ 7 9 ) . F597 National Cancer Institute, Carcinog. Tech. Rep. Ser., ~ (1979). F60] National Cancer Institute, Carcinogenesis Testing Program. DHHS Pub]. No. {NIH) 80-1754. F617 National Cancer Institute, Carcinogenesis Testing Program. DHHSPub]. No. (NIH) 80-1755. F62] A. Poland and E. Glover, Cancer. Res., 39, 3341 (1979). F637 J. DiGiovanni, A. Viaje, D.L. Berry, T . ~ Slaga, and M.R. Juchau, Bull. Environ. Contam. Toxicol., 18, 552 (1977). F647 G.M. Cohen-~-W.M. Bracken, R.P. Iyer, D.L.Berry, J.K. Selkirk, and T.J. Slaga, Cancer Res., 39, 4027 (]979). F65] J. DiG~vanni, D.L. Berry, G.L. Gleason, G.S. Kishore, and T.J. Slaga, Cancer Res., 40, 1580 (1980). F66] R.E. Kuori, T.H. Rude, R. Jonglekar, P.M. Dansette, D.M. Jerina, S.A. Atlas, I.S. Owens, and D.W. Nebert, Cancer Res., 38, 2777 (]978). F67] D.L. Berry, J. DiGiovanni, M.R?-Juchau, W.M. Bracken, G.L. Gleason, and T.J. Slaga, Res. Commun. Chem. Pathol. Pharmacol., 20, 101 {1978). F68] NTIS/PB 82-163684, Govt. Reports A~ouncements & Index, 11 (1982). F697 H.C. Pitot, T. Goldsworthy, H.A. Campbell, and A. Poland, Cancer Res., 40, 3616 (]980). F707 M.E. King, A.M. Shefner, and R.R. Bates, Environ. Hlth Perspec., 5, 163~1973). F717 J.G. Vos, J.G. Kreftenberg, H.W.B. Engel, A. Minderhoud and L.M. van Noorlejansen, Toxicology, 9, 75 (1978). F727 M.J. vanLogten, B.N. Gupta, E.E. McConnell, and J.A. Moore, Toxicology, I_5, 135 (1980). F737 J.G. Vos and J.A. Moore, Int. Arch. Allergy, 47, 77711974). F747 J.E. Thigpen, R.E. Faith, E.E. McConnell, and--~.A. Moore, Infect. Immun., 122, 1319 (1975). F757 J.G. Vos, J.A. Moore, and J.G. Zinkl, Environ. Hlth Perspec., 5, 149 (1973). F767 R.E. Faith, M.I. Luster, and J.A. Moore, Cell Immunol., 40, 27~ (1978). F77] A. Vecchi, A. Mantovani, M.Sironi, W. Luini, M. Cairo, a ~ S. Garattini, Chem.-Biol. Interact., 30, 337 (1980).