Hepatic Aryl Hydrocarbon Hydroxylase Research Articles

Cytotoxicity and mitochondrial dysfunction of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in isolated rat hepatocytes

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is an environmental contaminant that induces hepatic and extrahepatic oxidative stress and the mechanisms of TCDD-induced reactive oxygen species are not fully investigated. Moreover, the potential toxicity of TCDD in isolated rat hepatocytes is not fully explored. The aim of the current study was to explore the possible cytotoxic effect of TCDD on primary rat hepatocytes and to explore the impact of mitochondria in TCDD-induced toxicity. Hepatocytes were isolated from adult rat liver and incubated with 0, 5, 10 or 15 nM of TCDD for 24, 48 and 72 h. Cell viability, lactate dehydrogenase (LDH) leakage into media along with reactive oxygen species (ROS) generation and hydrogen peroxide (H 2O 2) production, mitochondrial membrane potential (Δψ m), superoxide dismutase (SOD), catalse (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), total thiol contents, hepatic aryl hydrocarbon hydroxylase (AHH), and ethoxyresorufin O-deethylase (EROD) were performed in hepatocytes. In addition, superoxide anion generation, lipid peroxidation (LPO), mitochondrial protein carbonyl content and respiratory chain complexes II and IV were assayed in hepatocyte mitochondria. Cell viability was significantly decreased while LDH leakage into media was significantly increased in a dose and time related manner. ROS generation and H 2O 2 production along with EROD and AHH activities were significantly increased in hepatocytes in the same pattern. The antioxidant enzymes SOD, CAT, GPx and GR and the non-enzymatic protein thiols, in addition to Δψ m were significantly decreased in hepatocytes in a concentration and time dependent pattern. On the other side, mitochondrial superoxide anion along with LPO and mitochondrial protein carbonyl content were significantly increased while the respiratory chain complexes II and IV activities were significantly decreased in hepatocyte mitochondria. This effect may lead to disruption in the functional integrity of hepatocytes and hepatocyte mitochondria. In conclusion, our data clearly show that TCDD induces hepatocyte toxicity and mitochondrial dysfunction by a mechanism involving generation of ROS. Mitochondria might be the primary source of (or at least contribute to) the oxidative stress response and resulting toxicological outcomes elicited by TCDD.

DOSE- AND TIME-DEPENDENT EFFECTS OF 2,3,7,8-TETRABROMODIBENZO-P-DIOXIN ON RAT LIVER

Dose- and time-dependent effects of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) on the liver were examined by single administration of TBDD by gavage to male and female rats. Fifteen Wistar rats of each sex per group received 0, 10, 30, 100 or 300 microg TBDD/kg body weight. Rats surviving to scheduled necropsy on Day 2, 7 or 36 after the TBDD administration were examined for hepatic histopathology, activities of hepatic microsomal enzymes and serum levels of lipids, total cholesterol and transaminases and hepatic concentrations of TBDD. Tigroid basophilic cytoplasm and hepatocellular hypertrophy were observed at 10 microg/kg on Day 2 or 7 through 36, whereas degenerative and aggressive lesions such as necrosis, fibrosis, multinucleated hepatocytes and disarrangement of hepatocytes occurred later at higher dose levels. Persistently increased activities of hepatic aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD), increased serum levels of total cholesterol and phospholipid and increased relative liver weight were observed in all groups dosed 10 mug/kg and above, suggesting that hepatic microsomal monooxygenases and basophilic cytoplasm of hepatocytes were early and sensitive indicators among those TBDD-induced effects. A dose-dependent increase in liver concentrations of TBDD on Day 2 was followed by logarithmic decreases in TBDD concentrations against the days elapsed after the TBDD administration. An elimination half-life (t(1/2)) of TBDD from the liver was estimated to range from 12 to 16 days. It was suggested that females were more susceptible to TBDD than males, and that acute hepatotoxicity of TBDD was as potent as that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Modifications in the carcinogen-metabolizing capacity of mouse liver treated with N-nitroso compounds.

One tenth of the LD50 as a single dose of various N-nitroso compounds (N-nitrosodimethylamine; NDMA, N-nitrosodiethylamine; NDEA, N-nitrosoethylpropylamine; NEPA, N-nitrosodipropylamine; NDPA, N-nitrosomethylethylamine; NMEA, N-nitroso-methylbutylamine; NMBA and N-nitrosoethylbutylamine; NEBA) was administrated into male mice. This dose markedly increased the hepatic contents of cytochrome P450 and cytochrome b5 and activities of NADPH-cytochrome c reductase and aryl hydrocarbon hydroxylase (AHH). The highest increase in the activity of cytochrome P450 (+142% relative to the control value) was shown in animals treated with either N-nitrosoethylpropylamine or N-nitrosodiethylamine. On the other hand, the lowest increase in the activity (+16%) was revealed in animals treated with N-nitrosodimethylamine (not significant compared to the control value). Cytochrome b5 content was increased by 190% of the control value in mice treated with N-nitrosomethylbutylamine, while N-nitrosodibutylamine induced the lowest increase (+20%). The maximum increase (+182%) in the activity of aryl hydrocarbon hydroxylase was shown in animals which received N-nitrosomethylbutylamine, while the lowest increase (+23%) in animals which received N-nitrosodiethylamine. The activity of hepatic AHH was also increased above the control value in animals treated with NDMA, NEBA NDPA, NMEA and NDBA by 138, 98, 90, 89 and 69%, respectively. Identically, NADPH-cytochrome c reductase activity was increased in animals which received NEPA, NMBA, NDMA, NMEA, NDPA, NEBA and NDEA by 202, 150, 110, 95, 94, 77 and 37%, respectively.

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.

Assessment of the toxic potential of PCB congener 81 (3,4,4′,5‐tetrachlorobiphenyl) to fish in relation to other non‐ortho‐substituted pcb congeners

AbstractPCB congener 81 (3,4,4′,5‐tetrachlorobiphenyl) has been detected in fish tissues from various sites in North America. The embryotoxicity of this compound to medaka (Oryzias latipes) and the induction of hepatic aryl hydrocarbon hydroxylase (AHH) in rainbow trout (Oncorhynchus mykiss) were determined to assess the toxic potency of this compound relative to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and two other non‐ortho‐substituted PCB compounds, congener 77 (3,3′,4,4′‐tetrachlorobiphenyl) and congener 126 (3,3′,4,4′,5‐pentachlorobiphenyl). The TCDD toxic equivalency factors (TEFs) estimated for congener 81 from two end points in the medaka embryotoxicity assay were 0.0014 (from mortality data) and 0.006 (from swim bladder inflation data). The TEF estimated for congener 81 from data on AHH induction in rainbow trout was 0.004. All TEFs were greater than those estimated for congener 77 but were less than the TEFs estimated for congener 126. On the basis of these toxicity data, it is suggested that this congener may contribute significantly to the toxic burden of planar halogenated aromatic hydrocarbons in fish.

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.

Polybrominated dibenzo-p-dioxins and dibenzofurans: literature review and health assessment.

Polybrominated dibenzo-p-dioxins (PBDDs) and dibenzofurans (PBDFs) occur as trace (ppb) contaminants in brominated flame retardants and are produced during combustion of these chemicals. They are also formed when organics are incinerated in the presence of bromine, e.g., in municipal and industrial incinerators and in internal-combustion engines. Combustion of organics in the presence of both bromine and chlorine results in the formation of mixed (i.e., bromo, bromo/chloro and chloro) halogenated dibenzo-p-dioxins and dibenzofurans (HDDs and HDFs). There are 4600 potential mixed congeners. The biological effects of PBDDs and PBDFs are similar, if not identical, to those of PCDDs and PCDFs. Both groups of compounds induce hepatic aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-o-deethylase (EROD) in rats and cause wasting and thymic atrophy in rats and guinea pigs. Tetrabrominated dinenzo-p-dioxin (TBDD) and dibenzofuran (TBDF) are reproductive toxins in mice and produce skin lesions in the rabbit-ear acnegenic test. The brominated compounds appear to bind to the same cytosolic receptors believed to mediate the toxicities of the chlorinated analogs. When compared on a molar-concentration basis, the brominated compounds are equipotent to the chlorinated analogs. TBDD is absorbed after oral, dermal, or intratracheal administration in rats, stored in the liver and adipose tissue, and eliminated in the feces through biliary excretion. The biological half-lives of the brominated compounds appear to be somewhat shorter than those of the corresponding chlorinated species. The brominated compounds, like their chlorinated congeners, have the potential to cause dermal, hepatic, and gastrointestinal toxicities in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Effects of bleached kraft mill effluent on fish in the St. Maurice River, Quebec

AbstractTissue levels of chlorinated dioxins and furans and the physiological and biochemical responses of fish were measured upstream and downstream of a bleached kraft mill in the St. Maurice River, Quebec. Relative to fish 10 km upstream of the source, white suckers (Catostomus commersoni) downstream showed evidence of chemical exposure, chemical metabolism, stress, and altered energy use. Levels of 2,3,7,8–tetrachlorodibenzo‐p‐dioxin and tetrachlorodibenzofuran in gutted whole fish averaged 23 and 260 pg/g immediately downstream of the plant and declined to 18 and 112 pg/g at 95 km downstream. The activity of hepatic aryl hydrocarbon hydroxylase (AHH) was 10X higher immediately downstream and 5X higher 95 km downstream. There were corresponding increases in relative liver size, hematocrit, serum glucose, serum protein, and fin‐ray asymmetries, with a pattern of response that matched enzyme induction. These responses pointed to a strong effluent effect and followed the patterns of tissue contamination by dioxins and furans and of water contamination by chlorinated phenolics and guaiacols. There were strong correlations between AHH induction and tissue contamination. Whereas lipid levels increased at all downstream sites, condition factor decreased at 95 km from the source, indicating that there may be disturbances in energy metabolism. Effects on sexual maturation, as shown by levels of serum hormones and gonad somatic index, were inconclusive.

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.