Hepatic Ethoxyresorufin O-deethylase Activity Research Articles

Comparative Ability of Various PCBs, PCDFs, and TCDD to Induce Cytochrome P450 1A1 and 1A2 Activity Following 4 Weeks of Treatment

Comparative Ability of Various PCBs, PCDFs, and TCDD to Induce Cytochrome P450 1A1 and 1A2 Activity Following 4 Weeks of Treatment. De Vito, M. J., Maier, W. E., Diliberto, J. J., and Birnbaum, L. S. (1993). Fundam. Appl. Toxicol. 20, 125-130. Toxic equivalency factors (TEFs) have been proposed for dibenzo- p-dioxins, dibenzofurans, and polyhalogenated biphenyls. The proposed toxic equivalency factors (TEFs), which are presently being evaluated in our laboratory, are currently used to estimate the potential health risk associated with exposure to complex mixtures containing these chemicals. In preliminary studies, equally potent doses, based on the published TEFs and relative enzyme-inducing potency, of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzofuran, 1,2,3,4,6,7,8,9-octachlorodibenzofuran, 3,4,3′,4′-tetrachlorobiphenyl, 2,3,4,3′,4′-pentachlorobipbenyl, 3,4,5,3′,4′-pentachlorobiphenyl, 2,3,4,3′,4′,5′-hexachlorobiphenyl, 2,3,4,5,3′,4′-hexachlorobiphenyl, and 3,4,5,3′,4′,5′-hexachlorobiphenyl were administered to female B6C3F1 mice 5 days a week over a 4-week period. Hepatic, skin, and lung cytochrome P450 1A1 and hepatic 1A2 activities were determined for all chemicals tested and compared to those from TCDD-treated mice. These initial studies indicate that the present TEFs do not reliably predict induction potency for many of the chemicals. Furthermore, our data suggest that the relative inductive potency of these chemicals may be tissue specific and that estimates of TEFs based on hepatic ethoxyresorufin O-deethylase activity may not accurately reflect the potency of these chemicals in nonhepatic tissue. The TEFs proposed for the "dioxin-like" polychlorinated biphenyls (PCBs) overestimate the potency of these compounds by factors of 10-1000. The present study indicates that more experimental data are required before TEFs for PCBs should be used in regulatory decision making.

Tissue distribution and induction of hepatic enzymes in rats after intravenous or subcutaneous administration of 2,3,7,8-TCDD

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) was administered intravenously (i.v.) to adult female Wistar rats as a single dose of 300 ng 14C-TCDD/kg body wt. At different time points after treatment (up to 175 days), TCDD concentrations were measured in different tissues and the activity of ethoxyresorufin O-deethylase (EROD) was measured in the liver microsomes. The results were compared with those observed after subcutaneous (s.c.) application of the same dose of TCDD. Highest TCDD concentrations in the liver (4.5 ± 1.1 ng/g wet wt) were measured 3 hours after i.v.-application, whereas after s.c.-administration of the same dose the maximum concentration in the liver (4.7 ± 0.9 ng/g wet wt) was reached 3 days after application. About 54% of the administered dose of TCDD was found in the whole liver 3 hours after i.v.-application. In adipose tissue, TCDD concentration was found to be maximal 7 days after the injection in both i.v.- and s.c.- treated groups (0.85 ± 0.06, and 0.82 ± 0.07 ng/g wet wt, respectively). Hepatic EROD activity was maximal 1 day after the i.v.-injection and 7 days after the s.c.-injection. EROD activities correlated well with target (hepatic) tissue concentrations of TCDD, regardless of the route of administration.

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.

Hepatic EROD activity in spot, Leiostomus xanthurus, exposed to creosote-contaminated sediments

Hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity was measured in spot, Leiostomus xanthurus, exposed in a laboratory flowthrough system to sediments contaminated with coal-tar creosote for 14 days. Mean total resolvable polycyclic aromatic hydrocarbon (PAH) concentrations tested were 16, 35, 76, 150, and 320 μg/liter. Fish at all test concentrations refused food; control fish fed normally. Severe fin erosion, epidermal lesions, and mortality were observed in fish exposed to 76, 150, and 320 μg/liter. EROD activity was dependent on creosote concentration and time of exposure. Median EROD activity in control fish was 62 pmol/min/mg protein ( N = 28). EROD activity in fish exposed to 35, 76, 150, and 320 μg/liter increased during the first 2 days of exposure and then declined. Maximal induction was almost 30-fold at 150 μg/liter. By day 7, EROD activity was not significantly different than that on day 0.

Induction of ethoxyresorufin- O-deethylase and inhibiyion of glucocorticoid receptor binding in skin and liver of haired and hairless HRS/J mice by topically applied 2,3,7,8-tetrachlorodibenzo- p-dioxin

The biochemical changes associated with the toxicity of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) have been reported to include alterations in glucocorticoid and epidermal growth factor receptors and mixed function oxidase (MFO) induction. TCDD induces MFO activity in skin of both haired and hairless HRS/J mice. However, epidermal hyperplasia and hyperkeratosis are produced only in the skin of hairless mice. Therefore, since steroid and growth factor responses are implicated in cell proliferation and differentiation, these mice constitute a model system for assessing the possible roles of glucocorticoid and epidermal growth factor receptors in the toxicity of TCDD. The effect of dermal TCDD application (12 μg/kg in 100 μl acetone) on ethoxyresorufin- O-deethylase (EROD) activity, glucocorticoid receptor binding and epidermal growth factor receptors in liver and skin of hairless and haired mice was determined. No differences existed in the basal number of cytosolic glucocorticoid receptors (B max) or the apparent equilbrium binding constants ( K d) in control liver, dorsal skin and abdominal skin of male and female hairless mice and haired male mice. Seven days after topical application of TCDD, decreases of approximately 38% were observed in the hepatic B max of the glucocorticoid receptors in both haired and hairless mice. However, in dorsal skin, TCDD decreased BP max by approximately 40% in hairless mice but only 18% in haired mice. The dexamethasone-glucocorticoid receptor complex from both liver and skin of control and TCDD treated mice had similar sedimentation co-efficients in sucrose density gradients. TCDD had no effect on the K d of glucocorticoid receptors of skin or liver in haired and hairless mice. No difference was observed in the time-dependent increases in hepatic EROD activity between haired and hairless mice after dermal application of TCDD. However, the maximum induction of EROD activity in microsomes from the skin of haired mice was only 60% of the activity observed in hairless animals. The induction of EROD by TCDD did not correlate temporally with the decrease in glucocorticoid receptor binding.

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.

Polychlorinated biphenyls concentration and cytochrome P-450E expression in winter flounder from contaminated environments

Reproductively mature winter flounder ( Pseudopleuronectes americanus) were collected from three Northeastern US sites with different degrees of polychlorinated biphenyl (PCB) and polynuclear aromatic hydrocarbon (PAH) contamination. Liver PCB concentrations (measured by capillary electron-capture gas chromatography) in fish collected in 1987 and 1988 at New Bedford Harbor (NBH) ranged from 7·4 to 191 μg/g dry wt; at Gaspee Point 3·9–17·7 μg/g; and at Fox Island 1·6–15·1 μg/g. Levels of ethoxyresorufin O-deethylase (EROD) activity were similar in fish of the same reproductive status from the three sites; however, immunoquantitated P-450E homolog (the EROD catalyst) content was significantly higher in NBH fish. This suggests that P-450E catalytic activity is being suppressed in the livers of the NBH animals. Recent studies in our laboratory indicate that competitive inhibition of P-450E catalytic activity by specific PCB congeners is one likely mechanism of this suppression. Hepatic EROD activity and P-450E content were significantly lower in gravid females (EROD, 0·10–0·69 units per nmol P-450; P-450E, 8·4–19% of spectral P-450) than in spent females (EROD, 1·94–3·49; P-450E, 48–109%), and ripe males (EROD, 1·86–3·41; P-450E, 48–84%) at all sites. This is consistent with a hormonal effect on P-450E expression, and thus EROD activity, in gravid females. The data indicate a complex relationship between levels of EROD activity, or P-450E, and tissue PCB concentrations in highly contaminated fish. How these variables are linked to altered endocrine or gonadal function is not yet known.

Induced cytochrome P-450 in Fundulus heteroclitus associated with environmental contamination by polychlorinated biphenyls and polynuclear aromatic hydrocarbons

Fundulus heteroclitus were collected from two sites in Rhode Island during the non-spawning season and analyzed for hepatic monooxygenase activities and for whole-body concentrations of polychlorinated biphenyls (PCB) and polynuclear aromatic hydrocarbons (PAH). Microsomal protein, total spectral cytochrome P-450 and cytochrome b 5 content did not differ between Seekonk River and Succotash Salt Marsh fish. Ethoxyresorufin O-deethylase (EROD) activity was significantly higher (3-fold) in Fundulus from the Seekonk River than in fish from Succotash Salt Marsh. Similarly, levels of the immunodetectable homolog of P-450E in Fundulus, a representative of the major PAH-inducible P-450 form (P450IA1) in teleosts, were higher in fish from the Seekonk River. In contrast, rates of aldrin epoxidase (AE) activity were the same in fish from the two sites. Concentrations of PCB were 1000-fold, and PAH 60-fold, greater in Seekonk River sediment than in Succotash Salt Marsh sediment. The bioavailability of these contaminants is not known, but the relative degree of contamination in the sediments is reflected in the fish tissues. Fish from both sites had very low concentrations of PAH in their tissues, representing as little as 0·1% of the concentration in the sediment, with tissue concentrations in Seekonk River fish exceeding those of Succotash fish by 4-fold. Polychlorinated biphenyl concentrations in Seekonk fish (1860 ± 176 ng per g dry weight) were 3-fold higher than in Succotash Marsh fish (636 ± 217 ng per g). These concentrations were nearly the same (Seekonk fish) or 300-fold greater (Succotash Marsh fish) than PCB concentrations in the surrounding sediment. The result is consistent with a rapid metabolism of PAH while PCBs appear to be slowly eliminated by these fish. Tissue PCB concentrations in Seekonk River fish were similar to those known to elicit strong P-450 induction in Fundulus. Thus, levels of hepatic P-450E and EROD activity, but not AE activity, reflect relative tissue concentrations of PCB and PAH in Fundulus from two sites. Liver hypertrophy was also observed in Seekonk River fish, indicating that altered liver function may have occurred in these animals.

Structure-activity relationships of dietary indoles: a proposed mechanism of action as modifiers of xenobiotic metabolism.

In an effort to understand the mechanism by which dietary indoles inhibit chemically initiated tumorigenesis in experimental animals, we have investigated the potency of 3-substituted and 1,3-disubstituted indoles on the induction of intestinal and hepatic cytochrome P-448-dependent monooxygenases in the rat. Oral intubation with indole-3-carbinol (13C), 1-methoxyindole-3-carbinol (N13C), 1-methoxyindole-3-carboxaldehyde (NCHO), and 3,3'-diindolylmethane (133') at 31 mumol/animal led to significant increases in hepatic ethoxyresorufin O-deethylase activity (EROD; 15, 7, 6, and 5-fold over control, respectively), while intubation with indole (IND), 3-methylindole (3MI), indole-3-carboxaldehyde (13CHO), and indole-3-acetonitrile (IAN) did not increase this monooxygenase activity over control levels. For the eight indoles tested, there was a strong relationship between instability in acidic solution, as indicated by the generation of insoluble products, and capacity to induce hepatic EROD. Further experiments indicated that 13C did not induce hepatic EROD when dosed ip (thus bypassing the acidity of the stomach). Acid treatment of 13C generated a reaction mixture (RXM) that induced EROD after ip or po dosing. Chromatographic fractionation of the RXM indicated that there exist at least four different 13C acid-condensation products in the RXM with the ability to induce EROD. The results presented strongly support the hypothesis that dietary indoles influence the levels of monooxygenase activities via a series of acid-condensation products generated upon introduction of the indole into the acidic environment of the stomach.

Induction of hepatic mixed function oxidase activity in trout ( Salvelinus fontinalis) by Aroclor 1254 and some aromatic hydrocarbon PCB replacements

Groups of brook trout ( Salvelinus fontinalis) were fed a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) or three PCB replacements based on phenylxylylethane or diisopropylnaphthalene mixtures at dose levels of 30 and 100 μg·g −1. Both dose levels of all the materials tested induced hepatic ethoxyresorufin O-deethylase activity; ethoxycoumarin O-deethylase activity was induced by Aroclor 1254 and one of the phenylxylylethane mixtures. Benzphetamine N-demethylase activity was not induced by any treatment. Effects on other indices of MFO induction, such as protein content and cytochrome P-450 content, were variable. The fish accumulated from 13 to 46% of the Aroclor 1254 dose but only 2 to 7% of the PCB replacements. All components of the phenylxylylethane mixtures were accumulated but some major components of the diisopropylnaphthalenes were not.