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2 mice
Immunopharmacology, 9 (1985) 155-164
Elsewer
155
IMO 00275
Immunosuppression by Polycyclic Aromatic Hydrocarbons: A Structure-Activity Relationship in B6C3F1 and DBA/2 Mice K i m b e r L. W h i t e Jr. 1,2, H e l e n H . L y s y 2 a n d M i c h a e l P. H o l s a p p l e 2 Departments of Pharmacology and Toxwology 2, and Bzostatistws 1, Medwal College of Vtrgmia, Richmond, VA 23298, U.S.A.
(Recewed 7 March 1984; accepted 19 February 1985)
Abstract: The structure-acttvlty relationship of polycychc aromatic hydrocarbon-induced lmmunosuppresslon was investigated usmg
the antibody-fornung cell response to sheep erythrocytes. Ten polycyclicaromatic hydrocarbons were evaluated following 14 days of subchromc exposure in female B6C3F1 mice Additionally, the lmmunotoxicltyof benzo(a)pyreneand 3 of its congeners was evaluated following acute exposure The ~mmunosuppresslonobserved following both subchromc and acute exposure was similar to the structure-activity relaUonship observed for the carcmogemcityof the compounds tested. Anthracene, chrysene, benzo(e)pyreneand perylene did not slgmficantly suppress the antibody-forming cell response compared to the corn oil vehicle controls. Benz(a)anthracene, benzo(a)pyrene, dlbenz(a,c)anthracene, and dxbenz(a,h)anthracenesuppressedthe antibody-formingcell response by 55 to 91%. The greatest suppression was observed with the 3-methylcholanthrene and 7,12,-dlmethylbenz(a)anthracene. Studies using mice with different susceptibility to aryl hydrocarbon hydroxylase mducUon demonstrated that susceptible m~ce(B6C3F1) were not as ~mmunosuppressed followmg exposure to polycychc aromatic hydrocarbons as were nonsuscepUblemice (DBA/2). Key words: Polycychc aromatic hydrocarbons, Immunosuppresslon; Structure-activity; Plaque-forming cells; Aryl hydrocarbon hy-
droxylase Introduction
The polycyclic aromatic hydrocarbons (PAHs) represent a class of ubiquitous contaminants which can enter the environment naturally during forest fires ( M c M a h o n and Tsoukalas, 1978) and through the decay of organic material (Ishiwatani and Takahisa, 1975). However, the major source of contamination occurs through the burning of fossil fuels, particularly coal, and from the exhaust products of internal combustion engines (Baum, 1978). The carcinogenicity of the P A H s has been extensively studied, as recently reviewed by Zedeck (1980). A clear structure-activity relationship exists with respect to carcinogenicity with this class of compounds. Slight variations in the number and arrangement of aromatic rings can lead to marked changes in carcinogenicity (Thakker et al., 1982). This structure-activity relationship has led to the
formulation of the 'bay-region theory' first proposed by Jerina and Daly (1976). This model attempts to identify which benzo-ring diol epoxide o f a given P A H will be the ultimate carcinogen and thus predicts relative carcinogenicity for a series of PAHs. The immunotoxicity of the P A H s has been known since 1952, when Malmgren et al. demonstrated that 3-methylcholanthrene, benz(a)anthracene, and dibenz(a,h)anthracene were capable of producing a depression in hemolysin titers against sheep erythrocytes. Using the antibody plaqueforming cell assay to sheep erythrocytes, Stjernsward (1966) verified these findings and further Abbrevtatlons AFC, antibody-forming cell; PAH, polycychc
aromatic hydrocarbon; AHH, aromatic hydrocarbon hydroxylase, TCDD, 2,3,7,8-tetrachlorodlbenzo-p-dloxm; TCDF, 2,3,7,8-tetrachlorodlbenzofuran; TCB, 3,4,Y,4'-tetrachlorobiphenyl, TPA, 12-o-tetradecanoylphorbol-13-acetate.
0162-3109/85/$03.30 © 1985 Elsevier Science Pubhshers B.V. (BiomedicalDivision)
156 demonstrated that noncarcinogenic hydrocarbons such as anthracene and benzo(e)pyrene had no immunosuppressive effects. While these early studies utilized acute exposure, Dean et al. (1983) have recently reported that subchronic administration of benzo(a)pyrene but not benzo(e)pyrene suppressed the humoral response. The results by Stjernsward (1966) and Dean et al. (1983) suggested that a similar structure-activity relationship exists for the immunotoxicity and carcinogenicity observed with the PAHs. The initial objective of the present investigation was to clarify the structure-activity relationship by measuring the effects of exposure to 10 PAHs on the IgM antibody response to sheep erythrocytes, which has recently been determined to be the most sensitive assay for measuring in vivo immunosuppression by PAHs (Dean et al., 1983; Munson et al., 1984). A second objective was to determine the relationship of the Ah (aromatic hydrocarbon) gene complex to the immunosuppression by the PAHs. It has been established that exposure to certain PAHs can induce drug metabolizing enzymes, particularly aromatic hydrocarbon hydroxylase (AHH) (Conney, 1982). In this regard the activity of the PAH is similar to that previously reported for other environmental contaminants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Poland and Glover, 1974) and the polychlorinated biphenyls (Poland and Glover, 1977). Utilizing strains of mice which vary in the inducibility of these enzymes, Silkworth and Grabstein (1982) and Vecchi et al. (1983) demonstrated that the immunosuppression of PCBs and TCDD, respectwely, was greatest in the most inducible strains. In the present investigation, selected PAHs were tested in B6C3F1 mice, a hybrid of C57BL/6 and C3H mice, which are both highly inducible strains, and DBA/2 mice, a highly resistant strain.
purchased from Litton (Frederick, MD, U.S.A.). Female DBA/2 mice were purchased from Charles River (Wilmington, MA). Animals arrived at 5-6 weeks of age and were quarantined for at least one week prior to use. Mice were randomly allocated (4 per cage) and were maintained on Lab Chow (Purina) and tap water ad libitum. Ambient temperatures were maintained at 21-24°C, relative humidity was controlled between 40 and 60%, and the light/ dark cycle was set at 12-h intervals.
Materials and Methods
Detection of antibody-forming cells
Animals
Splenic antibody-forming cells (AFC) were evaluated on the day of peak response, which occurred four days after immunization with sheep erythro-
Female (C57BL/6 x C3H)F1 (B6C3F1) mice were
Polycychc aromatic hydrocarbons PAHs were obtained from Aldrich Chemical Co. (Milwaukee, WI) and were of the highest purity available. Corn oil was obtained from Sigma Chemical Co. (St. Louis, MO). Chemicals were prepared weekly as solutions in corn oil. Dibenz(a,h) anthracene was prepared as a suspension in corn oil when its solubility was exceeded. During the dosing period, the solutions of test compounds were constantly stirred using a teflon-coated stir bar and magnetic stirrer.
Exposure Each mouse received a subcutaneous injection of 0.01 ml/g body weight of test compound or corn oll vehicle in the lower back region. The exposure volume was doubled when dibenz(a,h)anthracene was administered as a single injection. All compounds were administered on a #mol/kg basis. Depending on the experimental design, compounds were administered acutely (one injection) or subchronically (multiple injections). A variety of exposure regimens, which differed in the relationship between chemical exposure and antigen sensitization, were tested in B6C3F1 mice alone or in both B6C3F1 and DBA/2 mice. In comparison studies, both strains of mice were exposed concurrently.
157 cytes. AFC were enumerated using the hemolytic plaque technique as originally reported by Cunnlngham and Szenberg (1968) and recently described in an earlier investigation from this laboratory (Holsapple et al., 1984).
COMPOUND
STRUCTURE
ANTHRACENE
Statistwal analysis CHRYSENE
The data are presented as the mean -4- S.E. of the mean derived from 8 animals per group. When an analysis of variance showed significant differences, treatment groups were compared to the vehicle control group using the Dunnett's T test. Treatment groups differing from the control at the level of p < 0.05 or less were considered significant and are indicated by an asterisk.
BENZ(o)ANTHRACENE
DIBIENZ(o,c)ANTHRACENE
Results
Suppression of the IgM AFC response following exposure to polycyclic aromatic hydrocarbons The chemical structure of the PAHs evaluated for immunotoxicity are shown in Fig. 1. The immunosuppresslon resulting from daily exposure to PAHs for 14 days is shown in Table I. With the exception of dibenz(a,h)anthracene and benzo(a) pyrene, spleen weights from treated animals were not significantly different from their respective corn oil vehicle controls. A significant decrease in spleen cell number was observed in animals treated with dibenz(a,c)anthracene, dibenz(a,h)anthracene and 3-methylcholanthrene. The response, expressed either as IgM AFC/spleen or as IgM AFC/106 spleen cells, was not significantly affected by treatment with the simple PAHs, anthracene or chrysene. Similarly, there was no suppression observed following treatment with benzo(e)pyrene or perylene. Immunosuppression was observed with other PAHs containing 4 or 5 member rings. Treatment with benz(a)anthracene, dibenz(a,c)anthracene, and benzo(a)pyrene resulted in a ,-~ 60% suppression of the humoral immune response, while dibenz(a,h)anthracene suppressed the response by 91%. The greatest suppression was observed with the al-
DIBENZ(o,h)ANTHRACENE
BENZO(o)PYRENE
DtMETHYLBENZ(o~NTHRACENE
CHm
METHYLCHOLANTHRENE
BENZO(I)PYRENE
PERYLENE
® I~
Fig. 1 Chemical structures of polycychc aromatic hydrocarbons (PAHs) evaluated for immunotoxlclty
158 TABLE I The effects of subchromc exposure (14 day) to P A H s on IgM A F C response to sheep erythrocytes in B6C3F1 mice Treatment
Anthracene Chrysene Benz(a)anthracene DIbenz(a,c)anthracene Dlbenz(a,h)anthracene Benzo(a)pyrene 7,12,-Dlmethylbenz(a)anthracene 3-Methylcholanthrene Benzo(e)pyrene Perylene Vehicle (corn o11)
Dose ~mol/kg/day)
Spleen weight (mg)
Spleen cellularity ( x 10 ~)
IgM AFC/spleen ( x l0 s)
IgM A F C / 1 0 ~ spleen cells
(%)
160 160 160 160 160 160
145 130 114 96 63 I00
444444-
16.6 162 18.0 12 2 8.5 154
444444-
08 1.5 0.8 1.0' 04" 09
436 233 150 108 14 106
444444-
50 42 15" 25* 2* 21"
2581 1390 831 844 168 682
223 132 74* 147" 24* 119"
+ 37 a _ 26 a - - 56" -- 5 5 a --91 a _ 64 a
20 20 160 160
68 112 134 158 119 75 105 132
+
6
9.8
4-
1 0
47
4-
10 7 11 9 6 4 7
145 16.2 21 5 175 12.8 18.2 16.6
5: d: 44444-
1.0' 0.9 10 1.3 09 0.8 08
216 64 433 332 266 366 288
4444444-
11" 41" 24 32 50 37 55 32
458 4- 83* 1488 4- 270 1629:1:119 2030 4- 171 1879 4- 344 1935 4- 189 1932 4- 265 1695 4- 173
_ 76 b
4444444-
9 7 9 12 5* 8*
+ 44444-
Change
-23 ¢ _
4d
+ 8a
* p < 0.05. a-d Relative to 1st, 2nd, 3rd and 4th vehicle controls, respectwely
sure to selected PAHs did not affect final body weights, with the exception of perylene, which produced a significant increase (Table II). Dibenz(a, h)anthracene produced a 44% reduction in absolute thymus weight. None of the other PAHs produced any significant changes in thymus weight (Table II). The ~mmunosuppression of the IgM AFC re-
kyl-substituted PAHs. 7,12-Dimethylbenz(a)anthracene produced an equivalent degree of suppression (76%) at one-eighth the dose of the other PAHs. The same molar dose of 3-methylcholanthrene produced a 23% suppression which did not reach significance on a IgM AFC/106 spleen cells basis but was significantly different from the control when evaluated as AFC/spleen. Subchronic expoT A B L E II
The effects of subchromc exposure (14 days) to selected P A H s on body and t h y m u s weight m B6C3F1 mice Treatment
Dose ~mol/kg/day)
Body weight (g)
T h y m u s weight mg
Vehicle Anthracene Chrysene Benz(a)anthracene Dlbenz(a,c)anthracene Dlbenz(a,h)anthracene Benzo(a)pyrene Perylene *p<005
160 160 160 160 160 160 160
20 0 20.9 20.6 21 4 21 6 21 6 21.4 22.8
444444+ 4-
0.6 0.7 05 0.6 09 0.3 0.5 0 8*
68 78 76 69 58 45 69 77
% Body weight 44444444-
5 8 5 5 6 3* 5 4
0.35 0.37 0.37 0.32 0.26 0.21 0 32 034
+ 4444444-
0.03 0.04 0.03 0.02 0.02 0.02* 0.02 001
159 T A B L E III The effects of acute exposure of congeners of benzo(a)pyrene on the IgM A F C response to sheep erythrocytes m B6C3F1 mice Treatment a
Spleen weight (rag)
Spleen cellularity ( x I0 ~)
IgM AFC/spleen ( x l0 s)
IgM AFC/106 spleen ceils
Change (%)
Vehicle Dlbenz(a,c)anthracene Dibenz(a,h)anthracene Benzo(a)pyrene Perylene
83 77 71 76 99
15.5 13 3 10 5 12 5 16.5
227 63 46 34 298
1479 495 426 251 1741
-67 -71 -83 + 18
44444-
4 5 3 4 7
44444-
05 07 0.6* 0 8* 0.7
44444-
26 10" 18" 12" 47
4+ 444-
183 84* 151" 85* 240
a Each a m m a l recewed either vehicle (corn o11) or 1 m m o l / k g of the indicated isomer by subcutaneous rejection. * p < 0.05.
sponse following acute exposure was evaluated using benzo(a)pyrene and 3 of its congeners. Animals were injected subcutaneously with 1 mmol/kg of the test compounds and sensitized 5 days later with sheep erythrocytes. Four days post-sensitization, the number of AFC was determined. As shown in Table III, benzo(a)pyrene, dibenz(a,c)anthracene and dibenz(a,h)anthracene suppressed the AFC response, while perylene had no effect. The degree of suppression, which ranged from 67 to 83%, is sim-
ilar to the degree of suppression observed with subchronic administration (Table I). While spleen weights of treated animals showed no significant differences from control animals, both dibenz(a, h)anthracene and benzo(a)pyrene produced significant decreases in spleen cellularity. In a subsequent experiment, it was determined that the same dose of benzo(e)pyrene administered under the same regimen produced no suppression (data not shown).
T A B L E IV Effects of exposure to selected P A H s on IgM A F C response to sheep erythrocytes m B6C3F1 and DBA/2 mice Treatment a
Day(s) of treatment
D a y of sensmzatlon
Dally dose (#mol/kg)
B6C3F1
DBA/2
IgM AFC/10 ~ spleen cells Vehicle B(a)P Vehicle B(e)P DMBA Vehicle MCA
1-14 1-14 1-14 1-14 1-14 1-14 1-14
11 11 I1 11 11 11 I1
Vehicle B(a)P
I- 5 I- 5
2 2
160
Vehicle B(a)P
1 1
2 2
I000
160 160 20 20
1862 542 1935 1251 458 1932 1488
444+ 444-
240 130" 189 149 83* 265 270
Change (%)
IgM AFC/106 spleen cells
-23
1370 96 1340 1347 99 1782 901
2867 4- 147 2395 4- 291
-16
1790 4- 75 979 4- 109"
--45
1646 + 129 1593 4- 259
-
1642 4- 167 1254 4- 172
-24
-71 -35 -76
3
4+ 44444-
167 52* 85 70 23* 129 49*
Change (%)
-93 0 -93 -49
a Vehmle (corn o11), benzo(a)pyrene (B(a)P), benzo(e)pyrene (B(e)P, 7,12,-dlmethylbenz(a)anthracene ( D M B A ) and 3-methylcholanthrene (MCA). * p < 0.05
160
The role of genetic regulation on the suppression of the AFC response by polycyclic aromatic hydrocarbons The role of genetic regulation in the PAH-induced immunosuppression was evaluated in the B6C3F1 hybrid, an AHH-'responsive' mouse, and in the DBA/2 strain, the classic AHH 'non-responder' (Benedict et al., 1972). The results of these studies are summarized in Table IV. As can be seen in the upper section of Table IV both B6C3F1 and DBA/2 were suppressed by subchronic (14 day) exposure to benzo(a)pyrene, 7,12-dimethyl-benz(a)anthracene and 3-methylcholanthrene while benzo(e)pyrene failed to suppress the AFC response. However, with the three immunosuppressive PAHs, the degree of suppression was greatest in the DBA/2 mice. Benzo(a)pyrene was selected as the prototype for the PAHs and was subsequently tested under other exposure regimens. When the duration of the exposure to benzo(a)pyrene was decreased to 5 days (Table IV, second section) the magnitude of the suppression was also decreased. However, a marked difference between the mice was still observed, with the B6C3F1 animals showing a slight (16%), albeit nonsignificant, suppression, whde the DBA/2 animals were significantly suppressed (45%). When benzo(a)pyrene was injected acutely one day prior to sensitization (Table IV, third section), there were no significant differences between treated animals and their respective controls. The B6C3F1 animals were suppressed by 3% whde DBA/2 again showed a greater suppression of 24%.
Discussion The PAHs represent one of the most thoroughly studied classes of chemical compounds. They are ubiquitous environmental contaminants present in soil, air, foods and cigarette smoke. PAHs are biologically active compounds, in that most have been demonstrated to produce mutagenlcity and many are potent carcinogens (Zedeck, 1980). More recent studies have shown that several PAHs are also immunotoxic, when administered either acutely or
subchronically (Dean et al., 1983). Several studies have demonstrated that the humoral immune response is significantly suppressed following exposure to PAHs (Dean et al., 1983: Munson et al., 1984). An objective of the present investigation was to determine the structure-activity relationship of PAH-induced immunosuppression using the humoral antibody response to sheep erythrocytes and subchronic (14 day) exposure to 10 PAHs. The immunotoxicity of the PAHs administered subchronically for 14 days (Table I) or acutely (Table III) followed a structure-activity relationship. Animals treated with simple PAHs (anthracene and chrysene), while showing a slight depression of the AFC response, were not significantly different from animals treated with the corn oil vehicle. The 4- and 5-ring compounds, benz(a)anthracene, dibenz(a,c)anthracene, dibenz(a,h)anthracene and benzo(a)pyrene all produced a significant suppression of the AFC response which ranged from 55 to 91% of control. In contrast, benzo(e) pyrene and perylene failed to suppress the humoral immune response. Alkyl-substituted PAHs, 3methylcholanthrene and 7,12-dimethylbenz(a)anthracene produced a significant suppression of the AFC response when administered at one-eighth of the dose of the other PAHs evaluated. Based on these results, in which a decrease in the AFC response is used as an indicator of immunosuppresslon, the PAHs can be ranked in order of immunotoxic potency as follows: 7,12,-dimethylbenz(a)anthracene > 3-methylcholanthrene > dibenz(a,h)anthracene > benzo(a)pyrene f> benz(a)anthracene >~ dibenz(a,c)anthracene > chrysene > benzo(e)pyrene ~> perylene > anthracene. Although extensive research has been conducted on the structure-activity relationships of the PAHs, there are many problems associated with establishing a rank order with regard to carcinogenicity. The carcinogenic potency of PAHs can depend e.a. on the route of exposure, the tissue of comparison, and the test species and strain. Slaga et al. (1977) demonstrated that the carcinogenic potency of benzo(a)pyrene and its benzo-ring derivatives depended on the tumor model utilized. Benzo(a)pyrene showed greater carcinogenicity than the reactwe
161 metabolite (+)-B(a)P 7R,8S-diol-9S,10R-epoxide in the mouse skin model; however, the converse was true in studies in newborn mice. The structural orientation of the aromatic rings appears to be fundamental in determining the carcinogenic activity of this chemical class, since small variations in the arrangement and the number of rings lead to dramatic changes in carcinogenicity (Thakker et al., 1982). Benzo(a)pyrene, dibenz(a, c)anthracene, dibenz(a,h)anthracene, perylene and benzo(e)pyrene are all congeners of one another. Dibenz(a,c)anthracene has weak tumorigenic activity, whde benzo(a)pyrene and dibenz(a,h)anthracene are potent carcinogens (Cavalieri et al., 1978). The addition of alkyl substitutions to the benzanthracene moiety produces one of the most potent PAH carcinogens known (DlGiovannl et al., 1983). Neither benzo(e)pyrene nor perylene has been shown to be carcinogenic in the whole animal when administered alone (Horton and Christian, 1974; Chang et al., 1981). Benzo(e)pyrene has been shown to be carcinogenic when tested in the tumor initiation model in which the potent tumor promoter TPA was utilized. Perylene was found to have borderline initiating activity in this model (Scribner, 1973). Cavalieri et al. (1978) have compiled results from PAH studies which utilized various routes of exposure and tumor models to produce a relative ranking for carcinogenicity. Using their criteria, the PAHs we evaluated could be ranked in order of carcinogenic potency as follows: 7,12-dimethylbenz(a)anthracene > 3-methylcholanthrene t> benzo(a)pyrene > dibenz(a,h)anthracene > dibenz(a, c)anthracene > benz(a)anthracene ~> chrysene > benzo(e)pyrene = perylene = anthracene, which is essentially identical to the rank order of immunotoxic potency described above. In contrast with the definite structure-activity relationship for PAH-induced carcinogenicity, all of the PAHs we have evaluated in this investigation, with the exception of anthracene, have been reported to be mutagenic, following metabolic activation (Simmon, 1979; Wood et al., 1979). Several of the PAHs which did not decrease the AFC response, or only produced marginal suppression, have been shown to be highly mutagenic. Chrysene
was reported by McCann et al. (1975) to be a potent mutagen. Similarly, the noncarcinogenic congeners, benzo(e)pyrene and perylene, have been reported to be mutagenic in multiple strains of Salmonella typhimurium (Simmon, 1979). Perylene, when metabolically activated, was demonstrated to have mutagenic activity which was only slightly less than aflatoxin B1, one of the most potent mutagens known (Penman et al., 1980). Therefore, as regards two classical actions by the PAHs, immunotoxicity appears to be more closely related to carcinogenicity than to mutagenicity. The mechanism by which the PAHs suppress the AFC response remains to be elucidated. Nebert et al. (1975) have postulated the presence of a cytosolic receptor which specifically binds certain halogenated aromatic hydrocarbons, PAHs and polybrominated biphenyls. This cytosolic receptor, as well as other aspects of toxicity for these chemicals, may be mediated by the Ah gene complex (Poland and Glover, 1980). Vecchi et al. (1983) have studied the immunosuppressive effects of TCDD in five strains of mice, including the A H H 'nonresponder' strain, DBA/2, and the 'responder' strains, C57BL/6, C3H/HeN and their FI hybrid, B6C3F1. The B6C3F1 has been shown to have A H H inducibility which is between that of the C57BL/6 and C3H strains (Nebert et al., 1975). Vecchi's group showed that, following a single injection of TCDD (1.2 /zg/kg seven days prior to sensitization with sheep erythrocytes), the AFC response was significantly suppressed (27-40% of control) in the C57BL/6, C3H and B6C3F1 strains, while the DBA/2 animals treated with TCDD did not differ significantly from the DBA/2 control animals. Subchronic administration weekly for 8 weeks produced similar results when a dose of 0.5 #g/kg was utilized. 2,3,7,8-Tetrachlorodibenzofuran (TCDF) has been reported to be one of the most powerful competitors for the TCDD receptor (Greenlee and Poland, 1979). When this compound was tested in the C57BL/6 and DBA/2 strains, a dose of 6/~g/kg inhibited antibody production by 85% in the C57BL/6 mice, while only producing a 35% reduction in DBA/2 mice. Both TCDD and TCDF produced a significant decrease in thymus weights of
162 C57BL/6 animals, but failed to decrease the thymus weight in the DBA/2 mice. The planar polychlorinated biphenyl, 3,4,3',4'-tetrachlorobiphenyl (TCB) binds to the TCDD receptor and competes with TCDD for the cytosolic receptor in the C57BL/6 mouse (Poland and Glover, 1977). When TCB was administered two days prior to sensitization and two days after sensitization at a dose of 100 mg/kg, there was no suppression of the AFC response in the DBA/2 strain, while C57BL/6 animals were suppressed by 79% as compared to untreated controls (Silkworth and Grabstein, 1982). As with the study o f T C D D (Vecchi et al., 1983), thymus weights of TCDD-treated C57BL/6 animals were significantly decreased as compared to controls, while DBA/2 thymus weights were unaffected. Like TCB, TCDD and TCDF, the PAHs (benzo(a)pyrene, benz(a)anthracene, and 3-methylcholanthrene) have been shown to bind to the TCDD receptor and compete with TCDD at the receptor site (Poland et al., 1976). However, while TCB, TCDD and TCDF produced greater suppression in the humoral immune response in the inducible mice (C57BL/6, C3H and B6C3F1), the present investigation demonstrated that benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene all produced a greater suppression in the noninducible DBA/2 mice, regardless of the dosing regimen employed (Table IV). Similarly, TCB, TCDD and TCFD all produced significant suppression in thymus weight in C57BL/6 animals without altering weights in the DBA/2 strain. In our studies, no decrease in thymus weights of B6C3F1 mice occurred, with the exception of those animals treated with dibenzo(a,h)anthracene (Table II). These results cannot rule out the possibility that an interaction between PAHs and the TCDD receptor is associated with the observed immunosuppression. However, they do demonstrate that important differences exist in the immunotoxic profile of the PAHs when compared to the halogenated dioxins, furans and biphenyls. It is generally accepted that PAHs require metabolic activation to reactive metabolites for their mutagenic and carcinogenic effects (Sims, 1980).
Jerina and Daly's bay-region theory of PAH carcinogenesis was formulated from the results of biological studies on benzo(a)pyrene and its bay-region epoxides and the existing carcinogenicity data for alkyl- and fluoro-substituted benz(a)anthracenes (Miller and Miller, 1963). Recent work has definitively established that (-)-B(a)P (7R,8R)dihydrodiol is a proximate carcinogen and that (+)-B(a)P 7R,8S-diol-9S,10R-epoxide is the ultimate carcinogen derived from benzo(a)pyrene (Buening et al., 1978). These active metabolites are formed by the microsomal mixed function oxidase system, specifically through the actions of the cytochrome P450 and epoxide hydrase enzymes (Conney, 1982). Similar activations have also been postulated as a primary contributing factor to the carcinogenicity for dibenzanthracene and 3-methylcholanthrene (Thakker et al., 1982). As the structure-activity relationship for immunosuppression strongly correlated with the structure-activity relationship for carcinogenicity, one could conjecture that the same PAH metabolite mediates both responses. However, we have recently demonstrated that benzo(a)pyrene, but not benzo(e)pyrene, was capable of suppressing the in vitro AFC response to sheep erythrocytes (White and Holsapple, 1984). Suppression occurred when benzo(a)pyrene was directly added to spleen cell cultures without the presence of a metabolic activation system. Furthermore, when a metabolic activation system (S-9 microsomal proteins from induced B6C3F1 mice) was incorporated, the degree of suppression was not enhanced. Benz(a)anthracene, dibenz(a,h)anthracene, 7,12-dimethyldibenz(a)anthracene, and 3-methylcholanthrene produce a dose dependent suppression of the in vitro response when directly added to culture without a metabolic activation system (manuscript in preparation). In summary, the immunosuppression resulting from PAH exposure follows a structure-activity relationship which closely parallels the structureactivity relationship for carcinogenesis. Noncarcinogenic compounds failed to suppress the AFC response and, in some instances, enhanced the response. The alkyl-substituted PAHs, which are
163 a m o n g the m o s t p o t e n t carcinogens, p r o d u c e d the greatest i m m u n o s u p p r e s s i o n . A n intermediate degree of i m m u n o s u p p r e s s i o n is observed with those c o m p o u n d s which d e m o n s t r a t e i n t e r m e d i a t e carcinogenic potency. Studies using genetically different mice d e m o n s t r a t e that the p a t t e r n o f i m m u n o s u p p r e s s i o n observed with the P A H s is i n c o n sistent with that reported for h a l o g e n a t e d a r o m a t i c c o m p o u n d s which also b i n d to the T C D D cytosolic receptor. F u r t h e r studies are needed to determine the mechanism(s) by which P A H s p r o d u c e i m m u n o s u p p r e s s i o n . Specifically, the relationship between epoxides (i.e. the active carcinogenic m e t a b olites) a n d the decreased h u m o r a l i m m u n e response to sheep erythrocytes needs to be further investigated.
Acknowledgement This work was s u p p o r t e d by grants N I H ES-02520 a n d N01-ES-5001.
References Baum E (1978) Occurrence and surveillance of polycychc aromatic hydrocarbons. In Polycychc Hydrocarbons and Cancer, Vol 1 Eds H Gelbom and P Ts'O. New York: Academic Press, pp 45-70 Bene&ct W, Considme N, Nebert D (1972) Genetic differences m aryl hydrocarbon hydroxylase reduction and benzo(a)pyrene-produced tumorigenesis m the mouse Mol Pharmacol 6.266. Buenmg M, Wlslocki P, Levln W, Yagl H, Thakker D, Akagl H, Koreeda M, Jenna D, Conney A (1978) Tumongemclty of the optical enantlomers of the dmstereomenc benzo(a)pyrene 7,8-dlol-9,10-epoxldesm newborn mice" Exceptional actwity of (+)-7p,8ct-&hydroxy-9ct,10ct-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Proc Nail Acad Sci USA 75'5358 Buening M, Levin W, Wood A, Chang R, Lehr R, Taylor C, Yagl H, Jerina D, Conney A (1980) Tumorigenic activity of benzo(e)pyrene derivatives on mouse skin and in newborn mice. Cancer Res 40:203 Cavaherl E, Roth R, Grandjean C, Althoff J (1978) Mechanisms of tumor mltmtlon by polycychc aromatic hydrocarbons In Carcinogenesis, Vol. 3 Eds. P Jones and R Freudenthal. New York: Raven Press. pp. 273-284 Chang R, Levln W, Wood A, Lehr R, Kumar S, Yagi H, Jenna D, Conney A (1981) Tumongenicity of the dmstereomeric
bay-region benzo(a)pyrene 9,19-&ol-ll,12-epoxide in newborn mice Cancer Res 41 915. Conney A (1982) Induction of mlcrosomal enzymes by foreign chemicals and carcinogenesis by polycychc aromatic hydrocarbons Cancer Res 42:4875 CunnxnghamH, SzenbergA (1968) Further improvements m the plaqulng techmque for detecting single antibody-forming cells Immunology 14 599 Dean J, Luster M, Boorman G, Lauer L, Leubke R, Lawson L (1983) Selectwe lmmunosuppresslon resulting from exposure to the carclnogemccongener of benzopyrene m B6C3F1 m~ce Chn Exp Immunol 52 199. DiGlovanm J, Diamond L, Harvey R, Slaga T (1983) Enhancement of the skin tumor-m~tmtmg actw~ty of polycychc aromatic hydrocarbons by methyl-substitution at non-benzo 'bay-region' poslUons Carcmogenests 4 403 Greenlee W, Poland A (1979) Nuclear uptake of 2,3,7,8-tetrachlorodibenzo-p-dIoxinm C57BL/6J and DBA/2J mice Role of the hepatic cytosol receptor protein. J Btol Chem 254 9814 Holsapple M, Tucker A, McNerney P, White K (1984) Effects of N-Nltroso&methylamme on humoral lmmumty. J Pharmacol Exp Therap 229:493 Horton W, Christian G (1974) Cocarclnogemc vs incomplete carcmogemc actw~ty hydrocarbons Contrast between chrysene and benzo(b)tnphenylene. J Natl Cancer Inst 53 1017 Ishiwatam R, Takahlsa H (1975) Organic geochemistry of a 200-meter core sample from lake Biwa II Vertical dIstnbution of mono- and dlcarboxyhc acids and polynuclear aromatic hydrocarbons Proc Jpn Acad 51'436 Jerma D, Daly J (1976) Oxidation at carbon In Drug Metabohsm from Microbes to Man. Eds D Parker and R Smith. London.Taylor and Francis pp 13-32 Malmgren R, Bennlson B, McKinleyT (1952) Reduced antibody t~ters m m~ce treated w~th carcmogemc and cancer chemotherapeuUc agents. Proc Soc Exp Btol Med 79'484. McCann J, Chol E, Yamasakl E, Ames B (1975) Detection of carcinogens as mutagens in the Salmonella/mlcrosome test' Assay of 300 chemlcals. Proc Natl Acad Scl USA 72'5135. McMahon C, Tsoukalas S (1978) Polynuclear aromatic hydrocarbons in forest fire smoke In Carcmogeneszs, Vol 3 Eds P Jones and R Freudenthal New York. Raven Press. pp 61-73 Miller J, Miller E (1963) The carcinogemcitIes of fluoro denvatwes of 10-methyl-l,2-benzanthracene II Substitution of the K region and the 3', 6', and 7' positions. Cancer Res 23 229. Munson A, Butala J, Kuna R, Snyder N, Lysy H, White K (1984) Immunotoxtcolog~caleffects of subchromc exposure to benzopyrene m B6C3F1 male and female mice Toxicologzst 4.632. Nebert D, Robinson J, Niwa A, Kumakl K, Poland A (1975) Genetic expression of aryl hydrocarbon hydroxylase acUwty m the mouse. J Cell Pyszol 85'393 Penman B, Kaden D, Llber H, Skopek T, Thdly W, Hltes R (1980) Perylene is a more potent mutagen than benzo(a)pyr-
164 erie for S typhtmurtum. Mutatzon Res 77:271. Poland A, Glover E (1974) Comparison of 2,3,7,8-tetrachlorodibenzo-p-&oxm, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcolanthrene. Mol Pharmacol 10'349 Poland A, Glover E (1977) Chlorinated blphenyl reduction of aryl hydrocarbon hydroxylase activity" A study of the structure-activity relationship Mol Pharmacol 13 924. Poland A, Glover E (1980) 2,3,7,8-Tetrachloro&benzo-p-&oxm' Segregation of toxicity with Ah locus. Mol Pharmacol 17:86 Poland A, Glover E, Kende A (1976) Stereospeclfic, high affinity binding of 2,3,7,8-tetrachlorodlbenzo-p-dloxm by hepatic cytosol. Evidence that the binding species ~s receptor for reduction of aryl hydrocarbon hydroxylase. J Btol Chem 251'4936 Scnbner J (1973) Tumor lmtlatlon by apparently noncarcmogemc polycychc aromatic hydrocarbons. J Natl Cancer Inst 50 1717 Sllkworth J, Grabstem E (1982) Polychlorinated blphenyl lmmunotoxlcity dependence on isomer plananty and the Ah gene complex. Toxwol Appl Pharmacol 65' 109 Slmmon V (1979) In vitro mutagemclty assays of chemical carcmogens and related compounds with Salmonella typh,murtum. J Natl Cancer Inst 62.893 S~ms P (1980) The metabolic activation of chemical carcinogens Br Med Bull 36 11. Slaga T, Bracken W, Lewn W, Yagl D, Jenna D, Conney A (1977) Comparison of the tumor-lnmatmg activities of benzo(a)pyrene arene oxides and diolepoxldes., Cancer Res 37 4130
Stjernsward J (1966) Effects of noncarcmogemc and carcinogenic hydrocarbons on the antibody-forming cells measured at the cellular level in vitro J Natl Cancer Inst 36:1189 Thakker D, Yagl H, Nordqvlst M, Lehr R, Levm W, Wood A, Chang R, Conney A, Jerina D (1982) Polycychc aromatic hydrocarbons and carcinogenesis, the bay-region theory In Chemtcal Inductwn of Cancer, Vol IIA Eds J Acros, Y Woo, M Argus and D Lal New York: Academic Press. pp 727747 Vecchl A, Sirom M, Canegratl M, Recchla M, Garattim S (1983) Immunosuppresslve effects of 2,3,7,8-tetrachloro&benzo-p&oxm m strains of mice with different suscept~bdity to reduction ofaryl hydrocarbon hydroxylase. Toxicol Appl Pharmacol 68'434 White K, Holsapple M (1984) Direct suppression of the ,T: vitro antibody production by the carcinogen, benzo(a)pyrene, but not by the noncarcmogemc congener, benzo(e)pyrene. Cancer Res 44.3388 Wood A, Levm W, Chang R, Yagl H, Thakker D, Lehr R, Jerma D, Conney A (1979) Bay region actwatlon of carcmogemc polycychc hydrocarbons. In Polynuclear Aromatw Hydrocarbons Thtrd Internattonal Symposium on Chemtstry and Btology - - Carcmogenesis and Mutagenests Eds P Jones and P
Leber. Ann Arbor, Michigan Ann Arbor Science Pubhshers pp 531-551 Zedeck M (1980) Polycychc aromatic hydrocarbons, a review J Envtron Pathol Toxtcol 3:357
Elsewer
155
IMO 00275
Immunosuppression by Polycyclic Aromatic Hydrocarbons: A Structure-Activity Relationship in B6C3F1 and DBA/2 Mice K i m b e r L. W h i t e Jr. 1,2, H e l e n H . L y s y 2 a n d M i c h a e l P. H o l s a p p l e 2 Departments of Pharmacology and Toxwology 2, and Bzostatistws 1, Medwal College of Vtrgmia, Richmond, VA 23298, U.S.A.
(Recewed 7 March 1984; accepted 19 February 1985)
Abstract: The structure-acttvlty relationship of polycychc aromatic hydrocarbon-induced lmmunosuppresslon was investigated usmg
the antibody-fornung cell response to sheep erythrocytes. Ten polycyclicaromatic hydrocarbons were evaluated following 14 days of subchromc exposure in female B6C3F1 mice Additionally, the lmmunotoxicltyof benzo(a)pyreneand 3 of its congeners was evaluated following acute exposure The ~mmunosuppresslonobserved following both subchromc and acute exposure was similar to the structure-activity relaUonship observed for the carcmogemcityof the compounds tested. Anthracene, chrysene, benzo(e)pyreneand perylene did not slgmficantly suppress the antibody-forming cell response compared to the corn oil vehicle controls. Benz(a)anthracene, benzo(a)pyrene, dlbenz(a,c)anthracene, and dxbenz(a,h)anthracenesuppressedthe antibody-formingcell response by 55 to 91%. The greatest suppression was observed with the 3-methylcholanthrene and 7,12,-dlmethylbenz(a)anthracene. Studies using mice with different susceptibility to aryl hydrocarbon hydroxylase mducUon demonstrated that susceptible m~ce(B6C3F1) were not as ~mmunosuppressed followmg exposure to polycychc aromatic hydrocarbons as were nonsuscepUblemice (DBA/2). Key words: Polycychc aromatic hydrocarbons, Immunosuppresslon; Structure-activity; Plaque-forming cells; Aryl hydrocarbon hy-
droxylase Introduction
The polycyclic aromatic hydrocarbons (PAHs) represent a class of ubiquitous contaminants which can enter the environment naturally during forest fires ( M c M a h o n and Tsoukalas, 1978) and through the decay of organic material (Ishiwatani and Takahisa, 1975). However, the major source of contamination occurs through the burning of fossil fuels, particularly coal, and from the exhaust products of internal combustion engines (Baum, 1978). The carcinogenicity of the P A H s has been extensively studied, as recently reviewed by Zedeck (1980). A clear structure-activity relationship exists with respect to carcinogenicity with this class of compounds. Slight variations in the number and arrangement of aromatic rings can lead to marked changes in carcinogenicity (Thakker et al., 1982). This structure-activity relationship has led to the
formulation of the 'bay-region theory' first proposed by Jerina and Daly (1976). This model attempts to identify which benzo-ring diol epoxide o f a given P A H will be the ultimate carcinogen and thus predicts relative carcinogenicity for a series of PAHs. The immunotoxicity of the P A H s has been known since 1952, when Malmgren et al. demonstrated that 3-methylcholanthrene, benz(a)anthracene, and dibenz(a,h)anthracene were capable of producing a depression in hemolysin titers against sheep erythrocytes. Using the antibody plaqueforming cell assay to sheep erythrocytes, Stjernsward (1966) verified these findings and further Abbrevtatlons AFC, antibody-forming cell; PAH, polycychc
aromatic hydrocarbon; AHH, aromatic hydrocarbon hydroxylase, TCDD, 2,3,7,8-tetrachlorodlbenzo-p-dloxm; TCDF, 2,3,7,8-tetrachlorodlbenzofuran; TCB, 3,4,Y,4'-tetrachlorobiphenyl, TPA, 12-o-tetradecanoylphorbol-13-acetate.
0162-3109/85/$03.30 © 1985 Elsevier Science Pubhshers B.V. (BiomedicalDivision)
156 demonstrated that noncarcinogenic hydrocarbons such as anthracene and benzo(e)pyrene had no immunosuppressive effects. While these early studies utilized acute exposure, Dean et al. (1983) have recently reported that subchronic administration of benzo(a)pyrene but not benzo(e)pyrene suppressed the humoral response. The results by Stjernsward (1966) and Dean et al. (1983) suggested that a similar structure-activity relationship exists for the immunotoxicity and carcinogenicity observed with the PAHs. The initial objective of the present investigation was to clarify the structure-activity relationship by measuring the effects of exposure to 10 PAHs on the IgM antibody response to sheep erythrocytes, which has recently been determined to be the most sensitive assay for measuring in vivo immunosuppression by PAHs (Dean et al., 1983; Munson et al., 1984). A second objective was to determine the relationship of the Ah (aromatic hydrocarbon) gene complex to the immunosuppression by the PAHs. It has been established that exposure to certain PAHs can induce drug metabolizing enzymes, particularly aromatic hydrocarbon hydroxylase (AHH) (Conney, 1982). In this regard the activity of the PAH is similar to that previously reported for other environmental contaminants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Poland and Glover, 1974) and the polychlorinated biphenyls (Poland and Glover, 1977). Utilizing strains of mice which vary in the inducibility of these enzymes, Silkworth and Grabstein (1982) and Vecchi et al. (1983) demonstrated that the immunosuppression of PCBs and TCDD, respectwely, was greatest in the most inducible strains. In the present investigation, selected PAHs were tested in B6C3F1 mice, a hybrid of C57BL/6 and C3H mice, which are both highly inducible strains, and DBA/2 mice, a highly resistant strain.
purchased from Litton (Frederick, MD, U.S.A.). Female DBA/2 mice were purchased from Charles River (Wilmington, MA). Animals arrived at 5-6 weeks of age and were quarantined for at least one week prior to use. Mice were randomly allocated (4 per cage) and were maintained on Lab Chow (Purina) and tap water ad libitum. Ambient temperatures were maintained at 21-24°C, relative humidity was controlled between 40 and 60%, and the light/ dark cycle was set at 12-h intervals.
Materials and Methods
Detection of antibody-forming cells
Animals
Splenic antibody-forming cells (AFC) were evaluated on the day of peak response, which occurred four days after immunization with sheep erythro-
Female (C57BL/6 x C3H)F1 (B6C3F1) mice were
Polycychc aromatic hydrocarbons PAHs were obtained from Aldrich Chemical Co. (Milwaukee, WI) and were of the highest purity available. Corn oil was obtained from Sigma Chemical Co. (St. Louis, MO). Chemicals were prepared weekly as solutions in corn oil. Dibenz(a,h) anthracene was prepared as a suspension in corn oil when its solubility was exceeded. During the dosing period, the solutions of test compounds were constantly stirred using a teflon-coated stir bar and magnetic stirrer.
Exposure Each mouse received a subcutaneous injection of 0.01 ml/g body weight of test compound or corn oll vehicle in the lower back region. The exposure volume was doubled when dibenz(a,h)anthracene was administered as a single injection. All compounds were administered on a #mol/kg basis. Depending on the experimental design, compounds were administered acutely (one injection) or subchronically (multiple injections). A variety of exposure regimens, which differed in the relationship between chemical exposure and antigen sensitization, were tested in B6C3F1 mice alone or in both B6C3F1 and DBA/2 mice. In comparison studies, both strains of mice were exposed concurrently.
157 cytes. AFC were enumerated using the hemolytic plaque technique as originally reported by Cunnlngham and Szenberg (1968) and recently described in an earlier investigation from this laboratory (Holsapple et al., 1984).
COMPOUND
STRUCTURE
ANTHRACENE
Statistwal analysis CHRYSENE
The data are presented as the mean -4- S.E. of the mean derived from 8 animals per group. When an analysis of variance showed significant differences, treatment groups were compared to the vehicle control group using the Dunnett's T test. Treatment groups differing from the control at the level of p < 0.05 or less were considered significant and are indicated by an asterisk.
BENZ(o)ANTHRACENE
DIBIENZ(o,c)ANTHRACENE
Results
Suppression of the IgM AFC response following exposure to polycyclic aromatic hydrocarbons The chemical structure of the PAHs evaluated for immunotoxicity are shown in Fig. 1. The immunosuppresslon resulting from daily exposure to PAHs for 14 days is shown in Table I. With the exception of dibenz(a,h)anthracene and benzo(a) pyrene, spleen weights from treated animals were not significantly different from their respective corn oil vehicle controls. A significant decrease in spleen cell number was observed in animals treated with dibenz(a,c)anthracene, dibenz(a,h)anthracene and 3-methylcholanthrene. The response, expressed either as IgM AFC/spleen or as IgM AFC/106 spleen cells, was not significantly affected by treatment with the simple PAHs, anthracene or chrysene. Similarly, there was no suppression observed following treatment with benzo(e)pyrene or perylene. Immunosuppression was observed with other PAHs containing 4 or 5 member rings. Treatment with benz(a)anthracene, dibenz(a,c)anthracene, and benzo(a)pyrene resulted in a ,-~ 60% suppression of the humoral immune response, while dibenz(a,h)anthracene suppressed the response by 91%. The greatest suppression was observed with the al-
DIBENZ(o,h)ANTHRACENE
BENZO(o)PYRENE
DtMETHYLBENZ(o~NTHRACENE
CHm
METHYLCHOLANTHRENE
BENZO(I)PYRENE
PERYLENE
® I~
Fig. 1 Chemical structures of polycychc aromatic hydrocarbons (PAHs) evaluated for immunotoxlclty
158 TABLE I The effects of subchromc exposure (14 day) to P A H s on IgM A F C response to sheep erythrocytes in B6C3F1 mice Treatment
Anthracene Chrysene Benz(a)anthracene DIbenz(a,c)anthracene Dlbenz(a,h)anthracene Benzo(a)pyrene 7,12,-Dlmethylbenz(a)anthracene 3-Methylcholanthrene Benzo(e)pyrene Perylene Vehicle (corn o11)
Dose ~mol/kg/day)
Spleen weight (mg)
Spleen cellularity ( x 10 ~)
IgM AFC/spleen ( x l0 s)
IgM A F C / 1 0 ~ spleen cells
(%)
160 160 160 160 160 160
145 130 114 96 63 I00
444444-
16.6 162 18.0 12 2 8.5 154
444444-
08 1.5 0.8 1.0' 04" 09
436 233 150 108 14 106
444444-
50 42 15" 25* 2* 21"
2581 1390 831 844 168 682
223 132 74* 147" 24* 119"
+ 37 a _ 26 a - - 56" -- 5 5 a --91 a _ 64 a
20 20 160 160
68 112 134 158 119 75 105 132
+
6
9.8
4-
1 0
47
4-
10 7 11 9 6 4 7
145 16.2 21 5 175 12.8 18.2 16.6
5: d: 44444-
1.0' 0.9 10 1.3 09 0.8 08
216 64 433 332 266 366 288
4444444-
11" 41" 24 32 50 37 55 32
458 4- 83* 1488 4- 270 1629:1:119 2030 4- 171 1879 4- 344 1935 4- 189 1932 4- 265 1695 4- 173
_ 76 b
4444444-
9 7 9 12 5* 8*
+ 44444-
Change
-23 ¢ _
4d
+ 8a
* p < 0.05. a-d Relative to 1st, 2nd, 3rd and 4th vehicle controls, respectwely
sure to selected PAHs did not affect final body weights, with the exception of perylene, which produced a significant increase (Table II). Dibenz(a, h)anthracene produced a 44% reduction in absolute thymus weight. None of the other PAHs produced any significant changes in thymus weight (Table II). The ~mmunosuppression of the IgM AFC re-
kyl-substituted PAHs. 7,12-Dimethylbenz(a)anthracene produced an equivalent degree of suppression (76%) at one-eighth the dose of the other PAHs. The same molar dose of 3-methylcholanthrene produced a 23% suppression which did not reach significance on a IgM AFC/106 spleen cells basis but was significantly different from the control when evaluated as AFC/spleen. Subchronic expoT A B L E II
The effects of subchromc exposure (14 days) to selected P A H s on body and t h y m u s weight m B6C3F1 mice Treatment
Dose ~mol/kg/day)
Body weight (g)
T h y m u s weight mg
Vehicle Anthracene Chrysene Benz(a)anthracene Dlbenz(a,c)anthracene Dlbenz(a,h)anthracene Benzo(a)pyrene Perylene *p<005
160 160 160 160 160 160 160
20 0 20.9 20.6 21 4 21 6 21 6 21.4 22.8
444444+ 4-
0.6 0.7 05 0.6 09 0.3 0.5 0 8*
68 78 76 69 58 45 69 77
% Body weight 44444444-
5 8 5 5 6 3* 5 4
0.35 0.37 0.37 0.32 0.26 0.21 0 32 034
+ 4444444-
0.03 0.04 0.03 0.02 0.02 0.02* 0.02 001
159 T A B L E III The effects of acute exposure of congeners of benzo(a)pyrene on the IgM A F C response to sheep erythrocytes m B6C3F1 mice Treatment a
Spleen weight (rag)
Spleen cellularity ( x I0 ~)
IgM AFC/spleen ( x l0 s)
IgM AFC/106 spleen ceils
Change (%)
Vehicle Dlbenz(a,c)anthracene Dibenz(a,h)anthracene Benzo(a)pyrene Perylene
83 77 71 76 99
15.5 13 3 10 5 12 5 16.5
227 63 46 34 298
1479 495 426 251 1741
-67 -71 -83 + 18
44444-
4 5 3 4 7
44444-
05 07 0.6* 0 8* 0.7
44444-
26 10" 18" 12" 47
4+ 444-
183 84* 151" 85* 240
a Each a m m a l recewed either vehicle (corn o11) or 1 m m o l / k g of the indicated isomer by subcutaneous rejection. * p < 0.05.
sponse following acute exposure was evaluated using benzo(a)pyrene and 3 of its congeners. Animals were injected subcutaneously with 1 mmol/kg of the test compounds and sensitized 5 days later with sheep erythrocytes. Four days post-sensitization, the number of AFC was determined. As shown in Table III, benzo(a)pyrene, dibenz(a,c)anthracene and dibenz(a,h)anthracene suppressed the AFC response, while perylene had no effect. The degree of suppression, which ranged from 67 to 83%, is sim-
ilar to the degree of suppression observed with subchronic administration (Table I). While spleen weights of treated animals showed no significant differences from control animals, both dibenz(a, h)anthracene and benzo(a)pyrene produced significant decreases in spleen cellularity. In a subsequent experiment, it was determined that the same dose of benzo(e)pyrene administered under the same regimen produced no suppression (data not shown).
T A B L E IV Effects of exposure to selected P A H s on IgM A F C response to sheep erythrocytes m B6C3F1 and DBA/2 mice Treatment a
Day(s) of treatment
D a y of sensmzatlon
Dally dose (#mol/kg)
B6C3F1
DBA/2
IgM AFC/10 ~ spleen cells Vehicle B(a)P Vehicle B(e)P DMBA Vehicle MCA
1-14 1-14 1-14 1-14 1-14 1-14 1-14
11 11 I1 11 11 11 I1
Vehicle B(a)P
I- 5 I- 5
2 2
160
Vehicle B(a)P
1 1
2 2
I000
160 160 20 20
1862 542 1935 1251 458 1932 1488
444+ 444-
240 130" 189 149 83* 265 270
Change (%)
IgM AFC/106 spleen cells
-23
1370 96 1340 1347 99 1782 901
2867 4- 147 2395 4- 291
-16
1790 4- 75 979 4- 109"
--45
1646 + 129 1593 4- 259
-
1642 4- 167 1254 4- 172
-24
-71 -35 -76
3
4+ 44444-
167 52* 85 70 23* 129 49*
Change (%)
-93 0 -93 -49
a Vehmle (corn o11), benzo(a)pyrene (B(a)P), benzo(e)pyrene (B(e)P, 7,12,-dlmethylbenz(a)anthracene ( D M B A ) and 3-methylcholanthrene (MCA). * p < 0.05
160
The role of genetic regulation on the suppression of the AFC response by polycyclic aromatic hydrocarbons The role of genetic regulation in the PAH-induced immunosuppression was evaluated in the B6C3F1 hybrid, an AHH-'responsive' mouse, and in the DBA/2 strain, the classic AHH 'non-responder' (Benedict et al., 1972). The results of these studies are summarized in Table IV. As can be seen in the upper section of Table IV both B6C3F1 and DBA/2 were suppressed by subchronic (14 day) exposure to benzo(a)pyrene, 7,12-dimethyl-benz(a)anthracene and 3-methylcholanthrene while benzo(e)pyrene failed to suppress the AFC response. However, with the three immunosuppressive PAHs, the degree of suppression was greatest in the DBA/2 mice. Benzo(a)pyrene was selected as the prototype for the PAHs and was subsequently tested under other exposure regimens. When the duration of the exposure to benzo(a)pyrene was decreased to 5 days (Table IV, second section) the magnitude of the suppression was also decreased. However, a marked difference between the mice was still observed, with the B6C3F1 animals showing a slight (16%), albeit nonsignificant, suppression, whde the DBA/2 animals were significantly suppressed (45%). When benzo(a)pyrene was injected acutely one day prior to sensitization (Table IV, third section), there were no significant differences between treated animals and their respective controls. The B6C3F1 animals were suppressed by 3% whde DBA/2 again showed a greater suppression of 24%.
Discussion The PAHs represent one of the most thoroughly studied classes of chemical compounds. They are ubiquitous environmental contaminants present in soil, air, foods and cigarette smoke. PAHs are biologically active compounds, in that most have been demonstrated to produce mutagenlcity and many are potent carcinogens (Zedeck, 1980). More recent studies have shown that several PAHs are also immunotoxic, when administered either acutely or
subchronically (Dean et al., 1983). Several studies have demonstrated that the humoral immune response is significantly suppressed following exposure to PAHs (Dean et al., 1983: Munson et al., 1984). An objective of the present investigation was to determine the structure-activity relationship of PAH-induced immunosuppression using the humoral antibody response to sheep erythrocytes and subchronic (14 day) exposure to 10 PAHs. The immunotoxicity of the PAHs administered subchronically for 14 days (Table I) or acutely (Table III) followed a structure-activity relationship. Animals treated with simple PAHs (anthracene and chrysene), while showing a slight depression of the AFC response, were not significantly different from animals treated with the corn oil vehicle. The 4- and 5-ring compounds, benz(a)anthracene, dibenz(a,c)anthracene, dibenz(a,h)anthracene and benzo(a)pyrene all produced a significant suppression of the AFC response which ranged from 55 to 91% of control. In contrast, benzo(e) pyrene and perylene failed to suppress the humoral immune response. Alkyl-substituted PAHs, 3methylcholanthrene and 7,12-dimethylbenz(a)anthracene produced a significant suppression of the AFC response when administered at one-eighth of the dose of the other PAHs evaluated. Based on these results, in which a decrease in the AFC response is used as an indicator of immunosuppresslon, the PAHs can be ranked in order of immunotoxic potency as follows: 7,12,-dimethylbenz(a)anthracene > 3-methylcholanthrene > dibenz(a,h)anthracene > benzo(a)pyrene f> benz(a)anthracene >~ dibenz(a,c)anthracene > chrysene > benzo(e)pyrene ~> perylene > anthracene. Although extensive research has been conducted on the structure-activity relationships of the PAHs, there are many problems associated with establishing a rank order with regard to carcinogenicity. The carcinogenic potency of PAHs can depend e.a. on the route of exposure, the tissue of comparison, and the test species and strain. Slaga et al. (1977) demonstrated that the carcinogenic potency of benzo(a)pyrene and its benzo-ring derivatives depended on the tumor model utilized. Benzo(a)pyrene showed greater carcinogenicity than the reactwe
161 metabolite (+)-B(a)P 7R,8S-diol-9S,10R-epoxide in the mouse skin model; however, the converse was true in studies in newborn mice. The structural orientation of the aromatic rings appears to be fundamental in determining the carcinogenic activity of this chemical class, since small variations in the arrangement and the number of rings lead to dramatic changes in carcinogenicity (Thakker et al., 1982). Benzo(a)pyrene, dibenz(a, c)anthracene, dibenz(a,h)anthracene, perylene and benzo(e)pyrene are all congeners of one another. Dibenz(a,c)anthracene has weak tumorigenic activity, whde benzo(a)pyrene and dibenz(a,h)anthracene are potent carcinogens (Cavalieri et al., 1978). The addition of alkyl substitutions to the benzanthracene moiety produces one of the most potent PAH carcinogens known (DlGiovannl et al., 1983). Neither benzo(e)pyrene nor perylene has been shown to be carcinogenic in the whole animal when administered alone (Horton and Christian, 1974; Chang et al., 1981). Benzo(e)pyrene has been shown to be carcinogenic when tested in the tumor initiation model in which the potent tumor promoter TPA was utilized. Perylene was found to have borderline initiating activity in this model (Scribner, 1973). Cavalieri et al. (1978) have compiled results from PAH studies which utilized various routes of exposure and tumor models to produce a relative ranking for carcinogenicity. Using their criteria, the PAHs we evaluated could be ranked in order of carcinogenic potency as follows: 7,12-dimethylbenz(a)anthracene > 3-methylcholanthrene t> benzo(a)pyrene > dibenz(a,h)anthracene > dibenz(a, c)anthracene > benz(a)anthracene ~> chrysene > benzo(e)pyrene = perylene = anthracene, which is essentially identical to the rank order of immunotoxic potency described above. In contrast with the definite structure-activity relationship for PAH-induced carcinogenicity, all of the PAHs we have evaluated in this investigation, with the exception of anthracene, have been reported to be mutagenic, following metabolic activation (Simmon, 1979; Wood et al., 1979). Several of the PAHs which did not decrease the AFC response, or only produced marginal suppression, have been shown to be highly mutagenic. Chrysene
was reported by McCann et al. (1975) to be a potent mutagen. Similarly, the noncarcinogenic congeners, benzo(e)pyrene and perylene, have been reported to be mutagenic in multiple strains of Salmonella typhimurium (Simmon, 1979). Perylene, when metabolically activated, was demonstrated to have mutagenic activity which was only slightly less than aflatoxin B1, one of the most potent mutagens known (Penman et al., 1980). Therefore, as regards two classical actions by the PAHs, immunotoxicity appears to be more closely related to carcinogenicity than to mutagenicity. The mechanism by which the PAHs suppress the AFC response remains to be elucidated. Nebert et al. (1975) have postulated the presence of a cytosolic receptor which specifically binds certain halogenated aromatic hydrocarbons, PAHs and polybrominated biphenyls. This cytosolic receptor, as well as other aspects of toxicity for these chemicals, may be mediated by the Ah gene complex (Poland and Glover, 1980). Vecchi et al. (1983) have studied the immunosuppressive effects of TCDD in five strains of mice, including the A H H 'nonresponder' strain, DBA/2, and the 'responder' strains, C57BL/6, C3H/HeN and their FI hybrid, B6C3F1. The B6C3F1 has been shown to have A H H inducibility which is between that of the C57BL/6 and C3H strains (Nebert et al., 1975). Vecchi's group showed that, following a single injection of TCDD (1.2 /zg/kg seven days prior to sensitization with sheep erythrocytes), the AFC response was significantly suppressed (27-40% of control) in the C57BL/6, C3H and B6C3F1 strains, while the DBA/2 animals treated with TCDD did not differ significantly from the DBA/2 control animals. Subchronic administration weekly for 8 weeks produced similar results when a dose of 0.5 #g/kg was utilized. 2,3,7,8-Tetrachlorodibenzofuran (TCDF) has been reported to be one of the most powerful competitors for the TCDD receptor (Greenlee and Poland, 1979). When this compound was tested in the C57BL/6 and DBA/2 strains, a dose of 6/~g/kg inhibited antibody production by 85% in the C57BL/6 mice, while only producing a 35% reduction in DBA/2 mice. Both TCDD and TCDF produced a significant decrease in thymus weights of
162 C57BL/6 animals, but failed to decrease the thymus weight in the DBA/2 mice. The planar polychlorinated biphenyl, 3,4,3',4'-tetrachlorobiphenyl (TCB) binds to the TCDD receptor and competes with TCDD for the cytosolic receptor in the C57BL/6 mouse (Poland and Glover, 1977). When TCB was administered two days prior to sensitization and two days after sensitization at a dose of 100 mg/kg, there was no suppression of the AFC response in the DBA/2 strain, while C57BL/6 animals were suppressed by 79% as compared to untreated controls (Silkworth and Grabstein, 1982). As with the study o f T C D D (Vecchi et al., 1983), thymus weights of TCDD-treated C57BL/6 animals were significantly decreased as compared to controls, while DBA/2 thymus weights were unaffected. Like TCB, TCDD and TCDF, the PAHs (benzo(a)pyrene, benz(a)anthracene, and 3-methylcholanthrene) have been shown to bind to the TCDD receptor and compete with TCDD at the receptor site (Poland et al., 1976). However, while TCB, TCDD and TCDF produced greater suppression in the humoral immune response in the inducible mice (C57BL/6, C3H and B6C3F1), the present investigation demonstrated that benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene all produced a greater suppression in the noninducible DBA/2 mice, regardless of the dosing regimen employed (Table IV). Similarly, TCB, TCDD and TCFD all produced significant suppression in thymus weight in C57BL/6 animals without altering weights in the DBA/2 strain. In our studies, no decrease in thymus weights of B6C3F1 mice occurred, with the exception of those animals treated with dibenzo(a,h)anthracene (Table II). These results cannot rule out the possibility that an interaction between PAHs and the TCDD receptor is associated with the observed immunosuppression. However, they do demonstrate that important differences exist in the immunotoxic profile of the PAHs when compared to the halogenated dioxins, furans and biphenyls. It is generally accepted that PAHs require metabolic activation to reactive metabolites for their mutagenic and carcinogenic effects (Sims, 1980).
Jerina and Daly's bay-region theory of PAH carcinogenesis was formulated from the results of biological studies on benzo(a)pyrene and its bay-region epoxides and the existing carcinogenicity data for alkyl- and fluoro-substituted benz(a)anthracenes (Miller and Miller, 1963). Recent work has definitively established that (-)-B(a)P (7R,8R)dihydrodiol is a proximate carcinogen and that (+)-B(a)P 7R,8S-diol-9S,10R-epoxide is the ultimate carcinogen derived from benzo(a)pyrene (Buening et al., 1978). These active metabolites are formed by the microsomal mixed function oxidase system, specifically through the actions of the cytochrome P450 and epoxide hydrase enzymes (Conney, 1982). Similar activations have also been postulated as a primary contributing factor to the carcinogenicity for dibenzanthracene and 3-methylcholanthrene (Thakker et al., 1982). As the structure-activity relationship for immunosuppression strongly correlated with the structure-activity relationship for carcinogenicity, one could conjecture that the same PAH metabolite mediates both responses. However, we have recently demonstrated that benzo(a)pyrene, but not benzo(e)pyrene, was capable of suppressing the in vitro AFC response to sheep erythrocytes (White and Holsapple, 1984). Suppression occurred when benzo(a)pyrene was directly added to spleen cell cultures without the presence of a metabolic activation system. Furthermore, when a metabolic activation system (S-9 microsomal proteins from induced B6C3F1 mice) was incorporated, the degree of suppression was not enhanced. Benz(a)anthracene, dibenz(a,h)anthracene, 7,12-dimethyldibenz(a)anthracene, and 3-methylcholanthrene produce a dose dependent suppression of the in vitro response when directly added to culture without a metabolic activation system (manuscript in preparation). In summary, the immunosuppression resulting from PAH exposure follows a structure-activity relationship which closely parallels the structureactivity relationship for carcinogenesis. Noncarcinogenic compounds failed to suppress the AFC response and, in some instances, enhanced the response. The alkyl-substituted PAHs, which are
163 a m o n g the m o s t p o t e n t carcinogens, p r o d u c e d the greatest i m m u n o s u p p r e s s i o n . A n intermediate degree of i m m u n o s u p p r e s s i o n is observed with those c o m p o u n d s which d e m o n s t r a t e i n t e r m e d i a t e carcinogenic potency. Studies using genetically different mice d e m o n s t r a t e that the p a t t e r n o f i m m u n o s u p p r e s s i o n observed with the P A H s is i n c o n sistent with that reported for h a l o g e n a t e d a r o m a t i c c o m p o u n d s which also b i n d to the T C D D cytosolic receptor. F u r t h e r studies are needed to determine the mechanism(s) by which P A H s p r o d u c e i m m u n o s u p p r e s s i o n . Specifically, the relationship between epoxides (i.e. the active carcinogenic m e t a b olites) a n d the decreased h u m o r a l i m m u n e response to sheep erythrocytes needs to be further investigated.
Acknowledgement This work was s u p p o r t e d by grants N I H ES-02520 a n d N01-ES-5001.
References Baum E (1978) Occurrence and surveillance of polycychc aromatic hydrocarbons. In Polycychc Hydrocarbons and Cancer, Vol 1 Eds H Gelbom and P Ts'O. New York: Academic Press, pp 45-70 Bene&ct W, Considme N, Nebert D (1972) Genetic differences m aryl hydrocarbon hydroxylase reduction and benzo(a)pyrene-produced tumorigenesis m the mouse Mol Pharmacol 6.266. Buenmg M, Wlslocki P, Levln W, Yagl H, Thakker D, Akagl H, Koreeda M, Jenna D, Conney A (1978) Tumongemclty of the optical enantlomers of the dmstereomenc benzo(a)pyrene 7,8-dlol-9,10-epoxldesm newborn mice" Exceptional actwity of (+)-7p,8ct-&hydroxy-9ct,10ct-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Proc Nail Acad Sci USA 75'5358 Buening M, Levin W, Wood A, Chang R, Lehr R, Taylor C, Yagl H, Jerina D, Conney A (1980) Tumorigenic activity of benzo(e)pyrene derivatives on mouse skin and in newborn mice. Cancer Res 40:203 Cavaherl E, Roth R, Grandjean C, Althoff J (1978) Mechanisms of tumor mltmtlon by polycychc aromatic hydrocarbons In Carcinogenesis, Vol. 3 Eds. P Jones and R Freudenthal. New York: Raven Press. pp. 273-284 Chang R, Levln W, Wood A, Lehr R, Kumar S, Yagi H, Jenna D, Conney A (1981) Tumongenicity of the dmstereomeric
bay-region benzo(a)pyrene 9,19-&ol-ll,12-epoxide in newborn mice Cancer Res 41 915. Conney A (1982) Induction of mlcrosomal enzymes by foreign chemicals and carcinogenesis by polycychc aromatic hydrocarbons Cancer Res 42:4875 CunnxnghamH, SzenbergA (1968) Further improvements m the plaqulng techmque for detecting single antibody-forming cells Immunology 14 599 Dean J, Luster M, Boorman G, Lauer L, Leubke R, Lawson L (1983) Selectwe lmmunosuppresslon resulting from exposure to the carclnogemccongener of benzopyrene m B6C3F1 m~ce Chn Exp Immunol 52 199. DiGlovanm J, Diamond L, Harvey R, Slaga T (1983) Enhancement of the skin tumor-m~tmtmg actw~ty of polycychc aromatic hydrocarbons by methyl-substitution at non-benzo 'bay-region' poslUons Carcmogenests 4 403 Greenlee W, Poland A (1979) Nuclear uptake of 2,3,7,8-tetrachlorodibenzo-p-dIoxinm C57BL/6J and DBA/2J mice Role of the hepatic cytosol receptor protein. J Btol Chem 254 9814 Holsapple M, Tucker A, McNerney P, White K (1984) Effects of N-Nltroso&methylamme on humoral lmmumty. J Pharmacol Exp Therap 229:493 Horton W, Christian G (1974) Cocarclnogemc vs incomplete carcmogemc actw~ty hydrocarbons Contrast between chrysene and benzo(b)tnphenylene. J Natl Cancer Inst 53 1017 Ishiwatam R, Takahlsa H (1975) Organic geochemistry of a 200-meter core sample from lake Biwa II Vertical dIstnbution of mono- and dlcarboxyhc acids and polynuclear aromatic hydrocarbons Proc Jpn Acad 51'436 Jerma D, Daly J (1976) Oxidation at carbon In Drug Metabohsm from Microbes to Man. Eds D Parker and R Smith. London.Taylor and Francis pp 13-32 Malmgren R, Bennlson B, McKinleyT (1952) Reduced antibody t~ters m m~ce treated w~th carcmogemc and cancer chemotherapeuUc agents. Proc Soc Exp Btol Med 79'484. McCann J, Chol E, Yamasakl E, Ames B (1975) Detection of carcinogens as mutagens in the Salmonella/mlcrosome test' Assay of 300 chemlcals. Proc Natl Acad Scl USA 72'5135. McMahon C, Tsoukalas S (1978) Polynuclear aromatic hydrocarbons in forest fire smoke In Carcmogeneszs, Vol 3 Eds P Jones and R Freudenthal New York. Raven Press. pp 61-73 Miller J, Miller E (1963) The carcinogemcitIes of fluoro denvatwes of 10-methyl-l,2-benzanthracene II Substitution of the K region and the 3', 6', and 7' positions. Cancer Res 23 229. Munson A, Butala J, Kuna R, Snyder N, Lysy H, White K (1984) Immunotoxtcolog~caleffects of subchromc exposure to benzopyrene m B6C3F1 male and female mice Toxicologzst 4.632. Nebert D, Robinson J, Niwa A, Kumakl K, Poland A (1975) Genetic expression of aryl hydrocarbon hydroxylase acUwty m the mouse. J Cell Pyszol 85'393 Penman B, Kaden D, Llber H, Skopek T, Thdly W, Hltes R (1980) Perylene is a more potent mutagen than benzo(a)pyr-
164 erie for S typhtmurtum. Mutatzon Res 77:271. Poland A, Glover E (1974) Comparison of 2,3,7,8-tetrachlorodibenzo-p-&oxm, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcolanthrene. Mol Pharmacol 10'349 Poland A, Glover E (1977) Chlorinated blphenyl reduction of aryl hydrocarbon hydroxylase activity" A study of the structure-activity relationship Mol Pharmacol 13 924. Poland A, Glover E (1980) 2,3,7,8-Tetrachloro&benzo-p-&oxm' Segregation of toxicity with Ah locus. Mol Pharmacol 17:86 Poland A, Glover E, Kende A (1976) Stereospeclfic, high affinity binding of 2,3,7,8-tetrachlorodlbenzo-p-dloxm by hepatic cytosol. Evidence that the binding species ~s receptor for reduction of aryl hydrocarbon hydroxylase. J Btol Chem 251'4936 Scnbner J (1973) Tumor lmtlatlon by apparently noncarcmogemc polycychc aromatic hydrocarbons. J Natl Cancer Inst 50 1717 Sllkworth J, Grabstem E (1982) Polychlorinated blphenyl lmmunotoxlcity dependence on isomer plananty and the Ah gene complex. Toxwol Appl Pharmacol 65' 109 Slmmon V (1979) In vitro mutagemclty assays of chemical carcmogens and related compounds with Salmonella typh,murtum. J Natl Cancer Inst 62.893 S~ms P (1980) The metabolic activation of chemical carcinogens Br Med Bull 36 11. Slaga T, Bracken W, Lewn W, Yagl D, Jenna D, Conney A (1977) Comparison of the tumor-lnmatmg activities of benzo(a)pyrene arene oxides and diolepoxldes., Cancer Res 37 4130
Stjernsward J (1966) Effects of noncarcmogemc and carcinogenic hydrocarbons on the antibody-forming cells measured at the cellular level in vitro J Natl Cancer Inst 36:1189 Thakker D, Yagl H, Nordqvlst M, Lehr R, Levm W, Wood A, Chang R, Conney A, Jerina D (1982) Polycychc aromatic hydrocarbons and carcinogenesis, the bay-region theory In Chemtcal Inductwn of Cancer, Vol IIA Eds J Acros, Y Woo, M Argus and D Lal New York: Academic Press. pp 727747 Vecchl A, Sirom M, Canegratl M, Recchla M, Garattim S (1983) Immunosuppresslve effects of 2,3,7,8-tetrachloro&benzo-p&oxm m strains of mice with different suscept~bdity to reduction ofaryl hydrocarbon hydroxylase. Toxicol Appl Pharmacol 68'434 White K, Holsapple M (1984) Direct suppression of the ,T: vitro antibody production by the carcinogen, benzo(a)pyrene, but not by the noncarcmogemc congener, benzo(e)pyrene. Cancer Res 44.3388 Wood A, Levm W, Chang R, Yagl H, Thakker D, Lehr R, Jerma D, Conney A (1979) Bay region actwatlon of carcmogemc polycychc hydrocarbons. In Polynuclear Aromatw Hydrocarbons Thtrd Internattonal Symposium on Chemtstry and Btology - - Carcmogenesis and Mutagenests Eds P Jones and P
Leber. Ann Arbor, Michigan Ann Arbor Science Pubhshers pp 531-551 Zedeck M (1980) Polycychc aromatic hydrocarbons, a review J Envtron Pathol Toxtcol 3:357