Hepatic Aryl Hydrocarbon Hydroxylase Activity Research Articles

Toxicopathic hepatic lesions in subadult English sole ( pleuronectes vetuls) from Puget Sound, Washington, USA: Relationships with other biomarkers of contaminant exposure

Liver neoplasms are rarely detected in young wild fish. Therefore, other lesions occurring early in the histogenesis of hepatic neoplasia need to be considered as biomarkers of chemical contaminant exposure effects in monitoring studies, especially where adult fish are not available. Moreover, exposure effects may be more reliably assessed in younger fish that have not yet migrated extensively. Accordingly, livers of subadult English sole were histologically examined from nine sites in Puget Sound, WA and the same fish were assessed for contaminant exposure by measurement of fluorescent aromatic compounds (FACs) in bile, hepatic levels of polychlorinated biphenyls (PCBs) and cytochrome P4501A (CYP1A) as catalytic activity of aryl hydrocarbon hydroxylase (AHH), and Hydrophobic DNA adducts in liver by 32P-postlabelling. Although neoplasms were rare, higher prevalences of preneoplastic, regenerative, and unique degenerative/ necrotic lesions were detected in sole from contaminated sites. Prevalences of these early histopathologic biomarkers were significantly higher at the more contaminated sites, and concentration of mean biliary FACs at each capture site was a significant risk factor for most lesions, as determined by stepwise logistic regression. By this statistical method, we also demonstrated that several measures of bioaccumulation or biochemical response to contaminants were significant and near-significant risk factors for prevalences of most hepatic lesion categories. For example, mean hepatic AHH activity was a significant risk factor for prevalence of all lesion types, except neoplasms; hepatic PCB and xenobiotic-DNA adduct concentrations were significant risk factors for the most frequently detected lesion category, hepatocellular nuclear pleomorphism/megalocytic hepatosis, and the inclusive category ‘any early toxicopathic lesion’. These findings further support the utility of certain non-neoplastic liver lesions as early indicators of biological damage in subadult as well as adult fish exposed to xenobiotics in the marine environment.

Hepatic CYP1A in winter flounder ( Pleuronectes americanus) along the Northeast Coast: Results from the national benthic surveillance project

As part of the National Benthic Surveillance Project, cytochrome P4501A (CYP1A) and associated enzyme activities were measured in more than 700 samples of liver tissue collected from adult non-spawning winter flounder ( Pleuronectes americanus). Between 1988 and 1994, animals were sampled annually from 20 sites, ranging from Penobscot Bay in Maine to Great Bay, New Jersey. Analyses performed were assays of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufinO-deethylase (EROD) activities, and immunoquantitation of CYP1A by enzyme-linked immunosorbent assay (ELISA). From 1988 through 1990, CYP1A was measured by all three methods, to help determine an appropriate method for assessing temporal trends and to allow for eventual comparisons to methods used in other monitoring efforts. From 1991 through 1994, assays were only done for hepatic AHH activity. Fish from virtually all sites showed induction of hepatic AHH activity, consistent with earlier reports, though fish sampled from sites in the coastal waters of Maine generally showed the lowest mean AHH activities. At sites where fish were sampled during three to six separate years (15 of the 20), individual data were analyzed for monotonic temporal trends. There were trends towards increasing AHH activities over time in fish from the Raritan Bay/Long Island Sound area, and generally increasing trends for sites near Massachusetts (especially Boston Harbor) and Rhode Island. A decreasing trend was noted in fish from Great Bay, New Jersey, and there appeared to be decreasing activities in fish from the nearshore waters of Maine, though these trends were not statistically significant at the α = 0.05 level. The induction of CYP1A is strongly associated with exposure to chemical contaminants, and while the consequences of widespread and increasing induction of CYP1A are not known, these results suggest that the measurement of this enzyme system in benthic fish can be a useful tool for monitoring our coastal ecosystems.

EFFECTS OF p-CHLOROTOLUENE (PCT) ON RAT LUNG AND LIVER BENZO[a]PYRENE METABOLISM AND MICROSOMAL MEMBRANE STRUCTURE AND FUNCTION

Treatment of rats with p-chlorotoluene (PCT, 1 g/kg, ip) resulted in peak PCT blood and lung concentrations at 4 h, which declined to very low levels at 12 h. The concentration of PCT in the liver attained its highest value at 1 h and also declined to low levels at 12 h. In the dose-response study, PCT significantly decreased hepatic and pulmonary AHH activities at 0.5 g/kg, 1 h. Maximum inhibition was attained at 1 g/kg, 1 h, and further increase in the dose did not enhance the enzyme inhibition. In the time-course investigation, PCT (1 g/kg) maximally inhibited hepatic and pulmonary arylhydrocarbon hydroxylase (AHH) activities at 1 h and the decrease in enzyme activity was sustained through 12 h. Administration of PCT (1 g/kg, 1 h) also markedly decreased pulmonary cytochrome P-450 content, while hepatic cytochrome P-450 content was only slightly reduced. The partial decrease in cytochrome P450 content indicated altered levels of the P-450 isozymes, which may have profound effects on the metabolic disposition of benzo[a]pyrene [BaP]. BaP is regioselectively metabolized by two major isoforms of P-450 to toxic dihydrodiols and nontoxic phenol derivatives and there is a balance between these two metabolite groups. PCT (1 g/kg, 1 h) significantly inhibited the phenolic 3-OH BaP formation in both lung (52%) and liver (56%). The formations of BaP 7,8-dihydrodiol (146%) and 9,10-dihydrodiol (90%) were significantly elevated in the lung. The toxication to detoxication ratios were significantly elevated in both organs. Total quinone formation was markedly enhanced in the liver. Since PCT inhibited phenolic metabolite formation and increased dihydrodiol production, the activities of the isozymes that are responsible for their formations were determined. PCT (1 g/kg, 1 h) significantly inhibited cytochrome P-4502B1 in the lung (50%) and 2B1/2B2 in the liver (40%), while cytochrome P-4501A activity was not altered in either lung or liver. PCT increased phospholipid (PL) levels (45%) and conjugated diene (CD) formation (58%) in lung but not in liver, while membrane fluidity was increased [phospholipid/cholesterol (PL/CL) ratio; diphenylhexatriene (DPH) and trimethylammonium DPH (TMA-DPH) fluorescence polarization] in both organs. There was no apparent relationship between these membrane changes and alterations in MFO activity. Taken together the results suggest that PCT is capable of nonselectively inactivating the hepatic and pulmonary isozymes of P-450 that are responsible for the detoxication of BaP. The observed shift in the metabolism of BaP toward potentially more toxic metabolites suggests that concurrent exposure to BaP and PCT may result in greater toxicity, compared to exposure to BaP alone.

Immunosuppressive potential of several polycyclic aromatic hydrocarbons (PAHs) found at a Superfund site: new model used to evaluate additive interactions between benzo[ a]pyrene and TCDD

Exposure to environmental pollution is rarely limited to a single compound or even a single class of compounds. The Superfund site located in Massena, NY, is contaminated by both halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs). Since representatives of both HAHs and PAHs are capable of binding to the aromatic hydrocarbon receptor ( AhR), two well-documented AhR-mediated effects, immunosuppression and induction of hepatic aryl hydrocarbon hydroxylase (AHH) activity, were used to evaluate the individual and interactive toxicity of these compounds. Fifteen PAHs were first screened for their ability to suppress the antibody response in C57BL/6 ( Ah +/+) mice immunized 12 h after a single oral dose of 0.1, 1, 10, or 100 mg/kg. Acenaphthene, anthracene, benzo[ g, h, i]perylene, fluoranthene, fluorene, naphthalene, phenanthrene, and pyrene had little or no effect. Seven PAHs caused > 50% suppression at 100 mg/kg. Listed in order of decreasing potency they were benzo[ k]fluoranthene, benzo[ b]fluoranthene, indeno[1,2,3, c, d]pyrene, benzo[ a]pyrene, chrysene, dibenzo[ a, h]anthracene, and benz[ a]anthracene. Chrysene and benzo[ a]pyrene (B[ a]P), were further evaluated to determine the dependence of these effects on the Ah phenotype by comparing responses of C57BL/6 and congenic B6.D2 ( Ah −/−) mouse strains. Chrysene immunosuppression was maximal at 0.1 mg/kg and was Ah phenotype-independent whereas chrysene AHH induction was Ah phenotype-dependent, but a 100-fold less sensitive indicator of exposure. In contrast, B[ a]P immunosuppression and AHH induction were coincident in B6 mice and Ah phenotype-dependent. In the final phase, a new approach was used to evaluate toxic interactions. This approach considers the mechanism of action of each compound and accounts for the fact that the extent of increase in toxic response caused by an incremental change of dose is determined by its position on the dose-response curve rather than on the absolute amount of dose administered. Thus, the immunotoxic effects of combined exposure to B[ a]P and the AhR ligand, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), a representative HAH, were evaluated by combining the ED 20 of B[ a]P with the difference between the ED 20 and ED 40 of TCDD, and vice versa, to produce 40% suppression. The results of the combination were consistent with additivity regardless of the composite arrangement or phenotype although some antagonism could not be excluded with certainty.

Bioaccumulation of dietary 2,2′,4,4′,5,5′‐hexachlorobiphenyl and induction of hepatic arylhydrocarbon hydroxylase in rainbow trout (Oncorhynchus mykiss)

AbstractJuvenile rainbow trout (Oncorhynchus mykiss) were fed either 5 or 20 μg 2,2′,4,4′,5,5′‐hexachlorobiphenyl (245‐HxCB)/g diet (wet wt.) for 4, 8, or 12 weeks. Hepatic xenobiotic‐metabolizing enzyme activities and dietary 245‐HxCB accumulation in liver, muscle, and remaining carcass were determined. Liver‐to‐body weight ratios were not altered by either of the two 245‐HxCB concentrations. Relative growth rate increased with time but was not altered by 245‐HxCB concentration. Bioaccumulation of 245‐HxCB was dose and time dependent in all tissues without reaching apparent steady state. Hepatic arylhydrocarbon hydroxylase (AHH) activities increased with 245‐HxCB dose and with time. Ethoxyresorufin‐O‐deethylase (EROD) activities also increased in fish fed 20 μg 245‐HxCB/g diet. No 245‐HxCB‐induced changes in uridine diphosphoglucuronosyl transferase (UDP‐GT) or NADPH‐cytochrome‐c reductase (NCCR) activities were determined. High‐resolution GC‐MS analysis of the 245‐HxCB standard revealed trace (0.4‐0.5%) contamination by two mono‐ortho pentachlorobiphenyls (PnCBs): 2,3,3′,4,4′‐PnCB and 2,3,4,4′,5‐PnCB. Total liver accumulation of these contaminants was inversely related with corresponding EROD and AHH activities and estimated to contribute minimally to their induction. Results from this study suggested that long‐term dietary 245‐HxCB exposures induced cytochrome P4501A activities in rainbow trout liver.

Influence of growth hormone on the hepatic mixed function oxidase and transferase systems of rainbow trout

The effect of GH treatment on hepatic cytochrome P450 content, aryl hydrocarbon hydroxylase (AHH), aminopyrine-N-demethylase (AND), testosterone hydroxylase, testosterone 5α- and 5β-reductase, UDP-glucuronyl transferase (UDPGT) and glutathione S-transferase (GST) activities in immature rainbow trout were investigated. Hepatic cytochrome P450 content, AHH and GST activities were measured in both GH implanted and GH injected animals whereas other activities were assayed in GH implanted trout only.GH implants significantly decreased cytochrome P450 content at 15 days compared to the control but no significant effect was observed at 15 or 30 d when GH was injected biweekly. In both cases, AHH activity was significantly decreased by GH treatment compared to the control whereas GST remained unchanged. Compared to the control, GH implanted fish exhibited a pronounced inhibition of AND, a decreased 6β and 16β-testosterone hydroxylation, an inhibition of UDPGT with testosterone as substrate and an enhanced 17β-testosterone oxidation.

Indicators of reproductive development in prespawning female winter flounder (Pleuronectes americanus) from urban and non-urban estuaries in the northeast United States

The relationship between exposure to xenobiotic compounds and ovarian development was evaluated in prespawning female winter flounder (Pleuronectes americanus) sampled from eleven sites on the northeast coast of the United States during the 1988 and 1989 spawning seasons. Three sites were located in Boston Harbor, MA, four sites were in Raritan Bay, NJ and four sites were in nearby embayments. Sediments from these sites exhibited a wide range in concentrations of xenobiotic compounds (e.g. aromatic hydrocarbon (PAH) concentrations ranged from 20 to 50 000 ng/g dry wt. and polychlorinated biphenyl (PCB) concentrations ranged from 2 to 1400 ng/g dry wt.), with the sites in Boston Harbor and Raritan Bay the most heavily contaminated. The following parameters associated with ovarian development were measured: ovarian developmental stage, ovarian atresia, gonadosomatic index, plasma estradiol, fecundity and egg weight. Contaminant exposure was assessed by measuring concentrations of fluorescent aromatic compounds (FACs) in the bile, hepatic aryl hydrocarbon hydroxylase (AHH) activity, concentrations of PCB in liver, ovary and brain and concentrations of xenobiotic-DNA adducts in liver tissue. Additionally, liver tissue was examined histologically for the presence of suspected toxicopathic lesions. In general, indicators of contaminant exposure were elevated and prevalences of suspected toxicopathic lesions were highest in fish from sites within Boston Harbor and Raritan Bay. Hepatic AHH activity, however, was heavily influenced by the reproductive cycle and showed little correlation with other indicators of contaminant exposure. Evidence of decreased egg weight and increased atresia in fish with high tissue concentrations of PCB or high levels of FACs in bile was observed. However, contaminant exposure had no clear negative impact on gonadal recrudescence, gonadosomatic index, plasma estradiol concentrations, or fecundity in female winter flounder. These results are in contrast to results with another Pleuronectid species, English sole (Pleuronectes vetulus), which shows inhibited gonadal development and lower plasma estradiol concentrations at contaminated sites in Puget Sound, WA. The apparent difference between English sole and winter flounder in susceptibility to contaminant-induced reproductive dysfunction could be related to a number of factors, including the differences in the migratory behavior of the two species during the reproductive season. English sole reside in contaminated estuaries throughout vitellogenesis and move offshore to spawn, while winter flounder often remain offshore for extended periods during early vitellogenesis and move into contaminated estuaries prior to spawning. Because of these contrasting migration patterns, both the duration and timing of exposure to contaminants during gonadal recrudescence may differ substantially in these two species and may contribute, in part, to the observed differences in the prevalence of reproductive dysfunction in fish from contaminated estuaries.

Inhibition of rat hepatic aryl hydrocarbon hydroxylase activity by a series of 7-hydroxy coumarins: QSAR studies.

1. Molecular orbital calculations, by the Modified Intermediate Neglect of Differential Overlap (MINDO/3) method, of a series of twenty-five 8-acyl-7-hydroxy coumarins show that the inhibition of aryl hydrocarbon hydroxylase (AHH) activities (cytochrome P4501, CYP1 activity, primarily CYP1A1) for 23 of these compounds is related to their structural parameters. The two remaining compounds are the only chlorinated derivatives; these are inactive towards the AHH system and were excluded from the quantitative structure-activity relationship (QSAR) analysis. 2. The results of multiple regression analyses show that AHH activity is dependent on the energy of the highest occupied molecular orbital, E(HOMO), in a single variable expression for the 23 compounds. However, a three-variable expression involving superdelocalizabilities provides a more significant correlation with biological activity. 3. The inactivity of the two chlorinated derivatives can be rationalized in terms of their low degree of molecular planarity, as estimated by the area/depth2 parameter, which presumably precludes them from interaction with CYP1.

Mixed-function oxidase-specific activity in wild and caged speckled sanddabs Citharichthys stigmaeus in Elkhorn Slough, Moss Landing Harbor and nearshore Monterey Bay, California.

The goal of this study was to characterize the spatial, seasonal and annual hepatic activities of mixed-function oxidase (MFO) in the speckled sanddab Citharichthys stigmaeus, the most common fish in the Moss Landing area. In addition, techniques to monitor MFO activities in caged speckled sanddabs were developed and tested. Once the relationship between MFO activities in caged and wild fish populations is determined, caged fish could be used to monitor potential hydrocarbon impacts at Moss Landing, or other marine sites. During each of the spatial, seasonal and annual sediment samplings conducted in 1985-1987 as part of a separate hydrocarbon variability study at Moss Landing, 12 wild speckled sanddabs were collected from Moss Landing Harbor, Elkhorn Slough and nearshore Monterey Bay sites. In addition, four locations were chosen for a 14-day field caged fish experiment. The caged fish experiments successfully demonstrated the feasibility of using caged sanddabs as indicators of hydrocarbon exposure. The major source of variability in hepatic aryl hydrocarbon hydroxylase activity in wild speckled sanddabs from the Moss Landing area is due to seasonal rather than site differences. Significant relationships between caged fish MFO response and sediment hydrocarbon concentrations were found. It is possible that caged fish could be used in place of costly sediment sampling and analysis, and provide a more direct method to assess biological impacts. Many of the caging techniques demonstrated in this study can easily be transferred to other benthic flatfish, and other marine and freshwater environments.

Biomarkers of contaminant exposure in common terns and black‐crowned night herons in the Great Lakes

AbstractMorphological and biochemical indexes of contaminant exposure were examined in hatching common terns (Sterna hirundo) and black‐crowned night herons (Nycticorax nycticorax) from industrialized and nonindustrialized locations in the Great Lakes. In 1984, naturally incubated, pipping common tern and black‐crowned night heron embryos collected from industrialized locations exhibited smaller femur‐length‐to‐body‐weight ratios, elevated hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activities, and lower hepatic DNA concentrations (P < 0.05). In addition, a high incidence of subcutaneous edema was noted in pipping herons (P < 0.01). In 1985, reduced hatching success was observed for laboratory‐incubated common tern eggs collected from the industrialized sites, compared to nonindustrialized sites (P < 0.01). Day‐old hatchlings exhibited reduced femur‐length‐to‐body‐weight ratio, developmental anomalies, and elevated hepatic AHH activity (P < 0.05). For hatching common terns studied in 1984 and 1985, femur‐length‐to‐body‐weight ratio was inversely related to AHH activity (r = −0.67, P < 0.05) and inversely related to log‐transformed PCB concentrations (r = −0.70, P ≤ 0.05) of unincubated eggs from the same colony. The activity of AHH in hatching terns was also directly related (r = 0.71, P ≤ 0.05) to log‐transformed PCB concentrations in unincubated eggs. Other examined contaminants, including DDE, other organochlorine pesticides, and mercury, were not directly related to these effects.

Comparative Potencies of Aroclors 1232, 1242, 1248, 1254, and 1260 in Male Wistar Rats—Assessment of the Toxic Equivalency Factor (TEF) Approach for Polychlorinated Biphenyls (PCBs)

Comparative Potencies of Aroclors 1232, 1242, 1248, 1254, and 1260 in Male Wistar Rats—Assessment of the Toxic Equivalency Factor (TEF) Approach for Polychlorinated Biphenyls (PCBs). Harris, M., Zacharewski, T., and Safe, S. (1993). Fundam. Appl. Toxicol. 20, 456-463.Immature male Wistar rats were treated with several different doses of the commercial polychlorinated biphenyls (PCBs) Aroclors 1232, 1242, 1248, 1254, and 1260 (10, 40, 160, 480, and 2000 mg/kg) and the effects on body weight gain, thymic atrophy, and the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin O-deethylase (PROD) activities were measured 14 days after treatment. A significant inhibition in body weight gain was observed only in rats treated with high doses of Aroclors 1232 and 1248 and thymic atrophy was not observed for any of the Aroclors. All the Aroclors caused a dose-dependent increase in hepatic microsomal AHH, EROD, and PROD activities. The corresponding ED50 values for the induction of AHH-EROD activities varied from 51 to 678 mg/kg. Aroclor 1260 was the least active inducer of the P4501A1-mediated enzyme activities. In contrast, Aroclor 1260 was a potent inducer of PROD activity (ED50 37 mg/kg), but Aroclors 1232, 1242, 1248, and 1254 did not induce 50% of the maximal response at the highest dose used in this experiment 2000 mg/kg). Previous studies have quantitated the levels of those PCB congeners which induce AHH EROD activities in Aroclors 1232, 1242, 1254, and 1260 and their potencies or toxic equivalency factors (TEFs) relative to that of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) have also been estimated or experimentally determined. Using highly conservative TEF values it was demonstrated that the calculated ED50s for the Aroclors as inducers of AHH and EROD activity were significantly lower than the observed ED50 values. However, if the TEFs were derived directly from the relative potencies of the PCBs (compared to 2,3,7,8-TCDD) as inducers of AHH and EROD activity, then there was a good correspondence between the observed and calculated ED50 values.

BIOMARKERS OF CONTAMINANT EXPOSURE IN COMMON TERNS AND BLACK-CROWNED NIGHT HERONS IN THE GREAT LAKES

Morphological and biochemical indexes of contaminant exposure were examined in hatching common terns (Sterna hirundo) and black-crowned night herons (Nycticorax nycticorax) from industrialized and nonindustrialized locations in the Great Lakes. In 1984, naturally incubated, pipping common tern and black-crowned night heron embryos collected from industrialized locations exhibited smaller femur-length-to-body-weight ratios, elevated hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activities, and lower hepatic DNA concentrations (P < 0.05). In addition, a high incidence of subcutaneous edema was noted in pipping herons (P < 0.01). In 1985, reduced hatching success was observed for laboratory-incubated common tern eggs collected from the industrialized sites, compared to nonindustrialized sites (P < 0.01). Day-old hatchlings exhibited reduced femur-length-to-body-weight ratio, developmental anomalies, and elevated hepatic AHH activity (P < 0.05). For hatching common terns studied in 1984 and 1985, femur-length-to-body-weight ratio was inversely related to AHH activity (r = −0.67, P < 0.05) and inversely related to log-transformed PCB concentrations (r = −0.70, P ≤ 0.05) of unincubated eggs from the same colony. The activity of AHH in hatching terns was also directly related (r = 0.71, P ≤ 0.05) to log-transformed PCB concentrations in unincubated eggs. Other examined contaminants, including DDE, other organochlorine pesticides, and mercury, were not directly related to these effects.

Biochemical toxicology and disposition of Therminol 66 heat transfer fluid after inhalation or after dietary administration to male Sprague-Dawley rats.

The objectives of this study were to determine the disposition of Therminol 66 in rats and to determine the effects of this heat-transfer fluid on liver and kidney microsomal drug-metabolizing enzymes. Therminol 66 was administered to male Sprague-Dawley rats at various doses as either a single oral administration at 0, 100, or 300 mg/kg, or as a single 6-h inhalation exposure at 0 or 350 mg/m3. Animals were killed 48 h after gavage or after termination of inhalation exposure. Additional groups of animals were exposed to Therminol 66 via the diet at 0, 100, 500, or 5000 ppm for 14 d, or via repeated inhalation exposure at 0, 25, 250, or 1200 mg/m3 for 6 h/d for 14 d. These exposure scenarios represent approximately equivalent doses of Therminol 66 by the different routes of administration. No change in body weight was observed after acute oral or inhalation exposure, and little change in body weight was observed in animals administered Therminol 66 via the diet except at the highest dose. There was no change in kidney weight, and liver weights were increased only at the higher doses of Therminol 66. The body weight gain of animals exposed to Therminol 66 via inhalation decreased in a dose-dependent manner over the 2-wk exposure period. Results from the disposition study indicated that Therminol 66 did not appear to accumulate in the tissues examined and did not appear to be extensively absorbed after a single oral dose of 300 mg/kg. The whole-body elimination half-life was approximately 14 h and occurred primarily via the feces. There was no significant induction of hepatic aryl hydrocarbon hydroxylase (AHH) activity after single oral or inhalation exposures to Therminol 66. Ethoxycoumarin O-deethylase (ECOD) was significantly induced only in animals exposed to 350 mg/m3 via inhalation. Repeated dietary and inhalation exposures resulted in AHH and ECOD induction only at the highest doses, and the kidney appeared to be less sensitive than the liver. Animals exposed via inhalation demonstrated a greater hepatic inductive effect than did animals exposed via the diet, which may be due to absorption differences.(ABSTRACT TRUNCATED AT 400 WORDS)

Organ-selective induction of cytochrome P-450-dependent activities by indole-3-carbinol-derived products: Influence on covalent binding of benzo[ a]pyrene to hepatic and pulmonary DNA in the rat

Indole-3-carbinol (I3C) is a dietary modulator of carcinogenesis that can reduce the level of carcinogen binding to DNA. I3C-derived products are potent inducers of certain cytochrome P-450(CYP)-dependent enzyme activities. To investigate whether the protective effects of I3C against carcinogen damage to DNA are associated with increased activities of CYP1A1 enzymes, we examined the relationship of I3C-mediated organ-specific CYP enzyme induction with total levels of benzo[ a]pyrene (BP) binding to hepatic and pulmonary DNA of rats. Oral intubation (PO) of I3C (500 μmol/kg body wt.) in 10% DMSO in corn oil produced after 20 h, increases in ethoxyresorufin O-deethylase (EROD) activities (associated with CYP1A1 isozyme) of 700-fold, 245-fold and 36-fold in small intestine, lungs and liver, respectively, compared with activities in untreated controls. Hepatic aryl hydrocarbon hydroxylase (AHH) activity was increased 4-fold under these conditions. Pentoxyresorufin O-depentylase (PROD) activity (associated with CYP2B isozyme) was increased 6-fold in the liver but was unaffected in lung and small intestine. Intraperitoneal injection (IP) of I3C (500 μmol/kg body wt.) produced no significant change in EROD or PROD activities in lung, liver, or small intestine. PO administration of the acid reaction mixture (RXM) of I3C increased hepatic AHH activity (5-fold) and EROD activities in small intestine (650-fold), lung (100-fold) and liver (18-fold). IP administration of RXM (equivalent to 500 μmol I3C/kg body wt.) significantly increased only EROD activity in lung and liver, but did not affect EROD activity in small intestine, AHH activity in liver, or PROD activity in any of the organs examined. Twenty hours after inducer treatment, half of the rats were treated PO with 0.2 μmol [ 3H]BP in corn oil. Analysis of tissues 5 h after BP administration indicated that compared with untreated controls, administration of I3C and RXM by either route reduced by 30–50% the level of BP binding to hepatic DNA, an effect that was not correlated to CYP1A1 enzyme induction in any of the organs examined. However, PO administration of I3C and RXM produced a 50–70% decrease in carcinogen binding to pulmonary DNA, while IP administration of inducers had no effect on DNA binding in this organ. These results with the lung are consistent with an increased presystemic clearance of BP in the intestine and are discussed in terms of the role of induction of intestinal CYP1A1 activity in the decreased lymphatic and venous transport of unmetabolized BP to the lung.

Field studies of reproductive success and bioindicators of maternal contaminant exposure in English sole ( Parophrys vetulus)

Our studies have shown that early stages of the reproductive process in female English sole ( Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by measuring levels of polychlorinated biphenyls (PCBs) in ovarian and hepatic tissue, biliary levels of fluorescent aromatic compounds (FACs), and hepatic aryl hydrocarbon hydroxylase (AHH) activities. Additionally, the presence of significant liver disease or ovarian atresia was documented, and levels of plasma estradiol were measured. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining both egg and larval viability. Hepatic AHH activity showed a slight negative correlation with fertilization success, explaining only 4% of the variability. Increased levels of PCBs in ovaries of spawning female English sole were not associated with reduced reproductive success. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

Hepatic xenobiotic metabolizing enzymes in two species of benthic fish showing different prevalences of contaminant-associated liver neoplasms

English sole ( Parophrys vetulus) and starry flounder ( Platichthys stellatus) are closely related benthic fish which show substantial differences in prevalences of contaminant-associated hepatic neoplasms and putatively preneoplastic foci of cellular alteration when captured from estuaries containing a variety of organic chemical contaminants, including polycyclic aromatic hydrocarbons (PAH) and chlorinated hydrocarbons. Because PAH are strongly implicated as causative agents in the etiology of these lesions, several of the hepatic enzymes involved in activation and detoxication of PAH were studied in these two species. Hepatic aryl hydrocarbon hydroxylase (AHH), epoxide hydrolase (EH), and glutathione S-transferase (GST) activities were measured in animals sampled from both contaminated and reference areas. English sole, the species showing higher prevalences of contaminant-associated hepatic lesions, had higher (1- to 2-fold) hepatic activities of AHH and lower activities of EH (0.8-fold) and GST (1.8-fold) than those of starry flounder, regardless of site of capture. These results are largely consistent with increased activation and decreased detoxication of PAH by English sole in comparison to starry flounder. Both laboratory and field data suggested that the observed species differences in enzyme activities were constitutive and not related to differential exposure to contaminants. There were also substantial differences between these species with respect to expression of GST isoenzymes, in that starry flounder expressed two highly anionic GST isoenzymes which did not correspond to any GST isoenzymes expressed in English sole liver; a previous study in an elasmobranch fish showed that an anionic GST was most active toward PAH oxides. These differences in enzyme activities and isoenzyme profiles suggest a toxicological basis which may help to explain, at least in part, the differences in prevalences of contaminant-associated liver neoplasms between these two species.

Action of Caffeine in Altering the Carcinogen-Activating and -Detoxifying Enzymes in Mice.

Caffeine through drinking water, at low (100μg/ml), moderate (200 and 400μg/ml) and high (800μg/ml) doses, as well as by intragastric administration at 40 and 160mg/kg doses, increased the hepatic and pulmonary cytochrome P-450 (Cyt. P-450) levels in mice. The extent of elevation in the enzyme levels was dependent on the dose of caffeine. Maximum inducibility of hepatic arylhydrocarbon hydroxylase (AHH) was observed at the highest doses of caffeine by the two modes of treatment. However, the activities of AHH at the highest doses were not statistically different from the enzyme activities at the lowest doses. Caffeine treatment of animals through drinking water did not change the pulmonary AHH activity, whereas the intragastric administration of caffeine increased the enzyme activity in the lungs. Moreover, intragastric administration was more effective in inducing the hepatic AHH activity. Maximum increases in hepatic and pulmonary glutathione-S-transferase (GST), and in hepatic reduced glutathione (GSH), by the two modes of caffeine treatment of the animals was similar. The enhancement in AHH, Cyt. P-450, GST, and GSH may account for the anticarcinogenic activity of caffeine.

Hepatic activities of xenobiotic metabolizing enzymes and biliary levels of xenobiotics in english sole (Parophrys vetulus) exposed to environmental contaminants

English sole (Parophrys vetulus) are susceptible to the development of hepatic disease, including neoplasia, as a result of environmental exposure to polycyclic aromatic hydrocarbons (PAHs). The metabolism of PAHs, believed to be an essential factor in the development of neoplasia, has received considerable study in English sole, except that xenobiotic metabolizing enzymes (XMEs) have not been well-studied in this species. In the present work, the activities of hepatic aryl hydrocarbon hydroxylase (AHH), glutathione-S-transferase (GST), and epoxide hydrolase (EH) were measured in English sole exposed to several organic xenobiotics. These studies included an examination of the effects of captivity, the short-term responses of hepatic XME activities to several xenobiotic compounds, and detailed studies of the time- and dose-responses of hepatic XME activities to both a representative carcinogenic PAH (benzo[a]pyrene) and to a complex mixture of contaminants extracted from a sediment collected from a polluted area of Puget Sound, WA. Additionally, during the captivity and time- and dose-response studies, the levels of fluorescent aromatic compounds (FACs) were measured in the bile of the fish, both to provide an estimation of contaminant exposure and to evaluate the time- and dose-responses of this measure. The results of the captivity studies showed that the levels of FACs in bile were most affected by captivity, primarily as a result of changes in feeding status. The results of the exposure studies showed that xenobiotic metabolism, as reflected in hepatic activities of XMEs and levels of FACs in the bile, is altered by exposure to environmental contaminants. Whereas hepatic AHH activity could be rapidly and substantially increased by such exposure, activities of GST and EH were not affected, even up to 42 days after exposure. Moreover, because fish were exposed to a wide range of doses of chemicals or mixtures of chemicals which are known to be present in contaminated estuaries, and the responses of the hepatic AHH system and the levels of FACs in bile were measured at several time periods after exposure, the results provided substantial validation for the use of these two measures as bioindicators of exposure to environmental contamination in benthic fish.

Comparison of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐mediated hepatotoxicity in C57BL/6J and DBA/2J mice

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined by clinical chemistry and liver histopathology in Ah-responsive C57BL/6J (C57) and Ah-nonresponsive DBA/2J (DBA) mice. Hepatotoxicity was assessed at 1, 3, and 7 d following a single ip injection of TCDD at doses that maximally induce hepatic aryl hydrocarbon hydroxylase (AHH) activity (3 micrograms/kg for C57 and 30 micrograms/kg for DBA mice) and at doses approaching the LD50 (150 micrograms/kg for C57 and 600 micrograms/kg for DBA mice). Histological examination of liver sections was found to be a more sensitive detection method for TCDD-induced hepatic changes than clinical chemistry analyses. Dramatic differences in the development and type of liver injury were observed between TCDD-treated C57 and DBA mice. C57 mice given 3 micrograms TCDD/kg developed mild to moderate hepatic lipid accumulation in the absence of both inflammation and necrosis. Severe fatty change and mild inflammation and necrosis occurred in C57 mice that received 150 micrograms TCDD/kg. In contrast, DBA mice exposed to 30 micrograms TCDD/kg developed hepatocellular necrosis and inflammation without any fatty change. Only slight hepatic lipid accumulation occurred with some necrosis and inflammation in DBA mice given 600 micrograms TCDD/kg. The Ah locus may play a role in determining the sensitivity of C57 mice to the steatotic effects of TCDD.

Immunosuppressive and monooxygenase induction activities of polychlorinated diphenyl ether congeners in [formula omitted] mice: Quantitative structure-activity relationships

The dose-response effects of several polychlorinated diphenyl ether (polyCDE) congeners on the inhibition of the splenic plaque-forming cell (PFC) response to sheep red blood cell antigen and the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities were determined in male C57BL 6 mice. The immunotoxic potencies for the polyCDE congeners (ED50 values for the suppression of PFCs/spleen and PFCs/10 6 cells) followed the order 2,3,3′,4,4′,5-hexaCDE (0.5 and 2.2 μmol/kg)> 3,3′,4,4′,5-pentaCDE (8.8 and 5.1 μmol/kg) > 2,3′,4,4′,5-pentaCDE (21.8 and 14.2 μmol/kg) > 3,3′,4,4′-tetraCDE (50.6 and 28.7 μmol/kg) > 2,2′,4,4′,5,5′-hexaCDE (81.2 and 56.5 μmol/kg) > 2,2′,4,5,5′-pentaCDE (258 and 228 μmol/kg) > 2,2′,4,4′,5,6′-hexaCDE (>400 μmol/kg for both responses). The potencies of the polyCDE congeners as inducers of hepatic microsomal AHH and EROD activities were similar to their immunotoxicities and only one compound, namely, 2,3′,4,4′,5,5′-hexaCDE, did not cause dose-response immunosuppressive effects in the mice. The structure-activity relationships for the polyCDEs exhibited both differences and similarities. For example, the coplanar 3,3′,4,4′-tetraCDE and 3,3′,4,4′,5-pentaCDE congeners were less immunotoxic than their monoortho 2,3′,4,4′,5-pentaCDE and 2,3,3′,4,4′,5-hexaCDE analogs, respectively, and similar results were also observed for their enzyme induction potencies. For the corresponding polychlorinated biphenyls (PCB) congeners the coplanar compounds were significantly more active than their monoortho analogs. In addition, two diortho-substituted compounds, namely, 2,2′,4,5,5′-pentaCDE and 2,2′,4,4′,5,5′-hexaCDE, were also immunotoxic at a dose of 400 μmol/kg whereas, their PCB analogs were inactive. These studies clearly demonstrate that for the polyCDE congeners, increasing ortho-chloro substitution is less effective in reducing the activity of these congeners compared to the well-recognized ortho effects reported for the PCBs. The differences in the structure-activity relationships between polyCDEs and PCBs are related to the ether bridge in the polyCDEs in which the resultant increased bond length beweeen the two phenyl rings thereby diminishes the effects of ortho substituents on the biochemical and toxic potencies of these compounds.