Aryl Hydrocarbon Hydroxylase Activity Research Articles

Evaluation of toxic effects of petroleum hydrocarbon on Mytilus coruscus under different salinity based on an integrated biomarker response index

ABSTRACT An oxidative stress experiment of crude oil in Pinghu on Mytilus coruscus under different salinities (10, 15 and 20) for 15 days was carried out in this study. Oxidative stress of oil pollutants in digestive glands was measured by several typical biomarkers, the levels of ethoxyresourufin-O-deethylase (EROD) activities and aryl hydrocarbon hydroxylase (AHH) activities, CYP1A1mRNA content and DNA damage degree. The integrated biomarker response index (IBR) was introduced in for the quantitative evaluation of the responses of M. cephalus to the toxic effects of crude oil in Pinghu under different salinities. The results showed that the different salinities of crude oil in Pinghu can induce toxic effects on the digestive glands of M. coruscus to different extents, and the order of the degrees of toxicity, which were higher than those of the control groups, was 20 salinity > 15 salinity > 10 salinity. .

Erectile Dysfunction Drugs Changed the Protein Expressions and Activities of Drug-Metabolising Enzymes in the Liver of Male Rats.

Erectile dysfunction (ED) is a major health problem and is mainly associated with the persistent inability of men to maintain sufficient erection for satisfactory sexual performance. Millions of men are using sildenafil, vardenafil, and/or tadalafil for ED treatment. Cytochrome P450s (CYPs) play a central role in the metabolism of a wide range of xenobiotics as well as endogenous compounds. Susceptibility of individuals to the adverse effects of different drugs is mainly dependent on the expression of CYPs proteins. Therefore, changes in activities of phase I drug-metabolising enzymes [arylhydrocarbon hydroxylase (AHH), dimethylnitrosamine N-demethylase (DMN-dI), 7-ethoxycoumarin-O-deethylase (ECOD), and ethoxyresorufin-O-deethylase ((EROD)] and the protein expression of different CYPs isozymes (CYP1A2, CYP2E1, CYP2B1/2, CYP3A4, CYP2C23, and CYP2C6) were determined after treatment of male rats with either low or high doses of sildenafil (Viagra), tadalafil (Cialis), and/or vardenafil (Levitra) for 3 weeks. The present study showed that low doses of tadalafil and vardenafil increased DMN-dI activity by 32 and 23%, respectively. On the other hand, high doses of tadalafil, vardenafil, and sildenafil decreased such activity by 50, 56, and 52%, respectively. In addition, low doses of tadalafil and vardenafil induced the protein expression of CYP2E1. On the other hand, high doses of either tadalafil or sildenafil were more potent inhibitors to CYP2E1 expression than vardenafil. Moreover, low doses of both vardenafil and sildenafil markedly increased AHH activity by 162 and 247%, respectively, whereas high doses of tadalafil, vardenafil, and sildenafil inhibited such activity by 36, 49, and 57% and inhibited the EROD activity by 39, 49, and 33%, respectively. Low and high doses of tadalafil, vardenafil, and sildenafil inhibited the activity of NADPH-cytochrome c reductase as well as its protein expression. In addition, such drugs inhibited the expression of CYP B1/2 along with its corresponding enzyme marker ECOD activity. It is concluded that changes in the expression and activity of phase I drug-metabolising enzymes could change the normal metabolic pathways and might enhance the deleterious effects of exogenous as well as endogenous compounds.

Biomarker responses in the bivalve Chlamys farreri to the water-soluble fraction of crude oil

To investigate the effect of the water soluble fraction of crude oil (WSF) on marine bivalves, the scallop Chlamys farreri was exposed to three WSF concentrations (0.18 mg/L, 0.32 mg/L, and 0.51 mg/L, respectively) in seawater. Petroleum hydrocarbon contents in scallops and a suite of enzymes [7-Ethoxyresorufin-O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] in gills and digestive glands were monitored over 10 days. The results revealed that WSF affected the activity of the four enzymes in the gills and digestive glands. EROD activity in the gills was significantly induced in most individuals of the three test groups, while in the digestive gland it was significantly induced in the low-concentration group within 4 days but was inhibited in the middle- and high-concentration groups on days 1, 4, and 10. AHH activity in the gills of all treatment groups was significantly induced on day 1. In the digestive gland, AHH activity was induced in most individuals from the treatment groups. In all treatment groups, GST activity was significantly inhibited from days 2 to 10 in the gills and was induced after day 4 in the digestive gland. GPx activity in the gills was significantly inhibited throughout the exposure period in all treatment groups. There was no overall significant difference in GPx activity in the digestive gland between the control and treatment groups. Our results also revealed that petroleum hydrocarbon concentrations in the tissues increased linearly with exposure time. EROD activity in the digestive gland and GST and GPx activity in the gill tissue were negatively correlated with petroleum hydrocarbon body burden. These enzymes play important roles in detoxification and can act as potential biomarkers for monitoring petroleum hydrocarbon contaminants in the marine environment.

Curcumin and resveratrol in combination modulate drug-metabolizing enzymes as well as antioxidant indices during lung carcinogenesis in mice.

This study investigated combined chemopreventive potential of curcumin and resveratrol during benzo(a)pyrene (BP)-induced lung carcinogenesis in mice. The mice were segregated into five groups that included normal control, BP-treated, BP + curcumin-treated, BP + resveratrol-treated, and BP + curcumin + resveratrol-treated groups. A statistically significant increase in the levels of lipid peroxidation (LPO) was observed in the lungs of mice after 22 weeks of single dose of benzo(a)pyrene. Further, BP treatment also resulted in a significant increase in the enzyme activities of aryl hydrocarbon hydroxylase as well as drug-metabolizing enzymes, namely cytocrome P450 and cytochrome b5. On the other hand, reduced glutathione (GSH) levels, the activities of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione-S-transferase (GST) were found to be significantly decreased following BP treatment. Supplementation with curcumin and resveratrol to BP-treated mice significantly decreased the LPO levels, GSH levels, and enzyme activities of drug-metabolizing enzymes. Further, treatment of curcumin and resveratrol to BP-treated mice significantly elevated the activities of SOD, GR, and GST. Histoarchitectural studies showed well-differentiated signs of lung carcinogenesis following BP administration to mice. However, combined treatment with curcumin and resveratrol resulted in a noticeable improvement in the lung histoarchitecture. This study, therefore, concludes that curcumin and resveratrol when supplemented in combination regulate drug-metabolizing enzymes as well as antioxidant enzymes during lung carcinogenesis in mice.

The detoxification process, bioaccumulation and damage effect in juvenile white shrimp Litopenaeus vannamei exposed to chrysene

This study aimed to evaluate the effect of chrysene (CHR) on detoxification enzymes, bioaccumulation and effect of CHR on biomolecule damage in different organs of the juvenile white shrimp Litopenaeus vannamei. In this study, juvenile white shrimp L. vannamei were exposed to CHR for 21 days at four different concentrations as 0, 0.3, 2.1 and 14.7μg/L. Results showed that CHR bioaccumulation increased rapidly at first then reached a plateau. The activities of aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin O-deethylase (EROD), epoxide hydrolase (EH), glutathione-S-transferase (GST), sulfotransferase (SULT) and uridinediphosphate glucuronyltransferase (UGT) were induced and then became stable gradually. Moreover, 2.1 and 14.7μg/L CHR treatments increased activity of superoxide dismutase (SOD) in gills and hepatopancreas, while total antioxidant capacity (T-AOC) and GSH/GSSG were suppressed after CHR exposure. Additionally, lipid peroxidation (LPO) levels, protein carbonyl (PC) contents and DNA damage were induced throughout the exposure period, and different trends were detected with time of exposure. Overall, these novel findings of CHR bioaccumulation and resulted toxicity demonstrate that CHR could affect the physical status of L. vannamei. This study will form a solid basis for a realistic extrapolation scientific data for aquaculture water monitoring and food security.

Metabolites analysis, metabolic enzyme activities and bioaccumulation in the clam Ruditapes philippinarum exposed to benzo[a]pyrene

A study was performed on clams (Ruditapes philippinarum) exposed to 0.03, 0.3 and 3μg/L benzo[a]pyrene (B[a]P) for 21 days. B[a]P metabolite contents, activities of aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin O-deethylase (EROD), epoxide hydrolase (EH), dihydrodiol dehydrogenase (DD), glutathione-S-transferase (GST), sulfotransferase (SULT) and uridinediphosphate glucuronyltransferase (UGT) and B[a]P bioaccumulation were assayed in gills and digestive glands. Results showed that the order of B[a]P phase I metabolite contents was 9-hydroxy-B[a]P>B[a]P-1,6-dione>B[a]P-7,8-dihydrodiol, and the concentration of B[a]P-7,8-dihydrodiol sulfate conjugates was higher than that of B[a]P-7,8-dihydrodiol glucuronide conjugates. B[a]P accumulation and the activities of AHH, EROD, EH, DD, SULT and UGT increased first and then reached equilibrium. GST activity was induced first and then depressed. The concentration of B[a]P was far higher than that of its metabolites. Besides, there were no significant differences between enzyme activities in gills and those in digestive glands. These results provided information on B[a]P metabolic mechanism in bivalve and scientific data for pollution monitoring and food security.

Mitochondrial Targeting of Cytochrome P450 (CYP) 1B1 and Its Role in Polycyclic Aromatic Hydrocarbon-induced Mitochondrial Dysfunction

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41-48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.

A physiologically based toxicokinetic and toxicodynamic model links the tissue distribution of benzo[a]pyrene and toxic effects in the scallop Chlamys farreri

A physiologically based toxicokinetic and toxicodynamic (PBTK-TD) model was developed for benzo[a]pyrene (B[a]P) in scallop Chlamys farreri. The PBTK model structure consisted of gill, digestive gland, adductor muscle, hemolymph and other tissues. In TD modeling, aryl hydrocarbon hydroxylase (AHH) activity assay, comet assay, protein carbonyl measurement and lipid peroxidation level determination in digestive gland were used as biomarkers to reflect toxic effects. We integrated B[a]P concentration and biomarkers by using sigmoid Emax model in digestive gland. The PBTK-TD model predicted the B[a]P concentrations within each organ compartment and the toxic effects in digestive gland. The results showed that the predicted and measured data in different organs were in good agreement and comet assay was considered as the best biomarker. This model would serve as a useful tool for pollution monitoring and food security.

Application of the biomarker responses in scallop (Chlamys farreri) to assess metals and PAHs pollution in Jiaozhou Bay, China

Scallops (Chlamys farreri) were collected at three sites in the Jiaozhou Bay, China in April and July 2007. The responses of biomarkers were evaluated for assessment of physiological status of scallops and pollution in the areas. Compared with site S1, the activity of aryl hydrocarbon hydroxylase was induced notably (P < 0.05) and glutathione S-transferase activity was inhibited significantly (P < 0.05) in S2 and S3. The levels of DNA alkaline unwinding, DNA protein crosslinks, protein carbonyl and lipid peroxidation were tested and indicated the oxidative stress situation of scallops. There was significant difference of biomarker levels between sampling seasons (P < 0.05), and between digestive gland and gill (P < 0.05). The results provided the reference data for multiple-pollution assessment in the marine environment and indicated that tissue type and seasons affect biomarkers, therefore these factors should be taken into consideration when biomarkers are used for environmental assessment purposes.

Biomarkers and bioaccumulation of clam Ruditapes philippinarum in response to combined cadmium and benzo[α]pyrene exposure

Biochemical and molecular biomarkers (the contents of metallothionein (MT), glutathione (GSH), the activities of aryl hydrocarbon hydroxylase (AHH), glutathione S-transferase (GST) and superoxide dismutase (SOD) and the mRNA expressions of GST-pi and Cu, Zn-SOD) were evaluated in clams Ruditapes philippinarum exposed to cadmium (Cd, 15μg/L) and benzo[α]pyrene (BaP, 0.01μg/L) individually and in combination (15μg/L Cd+0.01μg/L BaP) for 21days. The accumulation of Cd, BaP and the biomarkers measured in the gills and digestive glands of the clam showed significant increase in combination treatment and it was significantly higher than the Cd or BaP treatment (P>0.05). The contents of MT increased in Cd and Cd+BaP treatment, while AHH activities were increased in Bap and Cd+BaP treatment (P>0.05). GSH levels enhanced in Cd group and declined significantly in Cd+BaP treatment (P>0.05). The activities of GST, SOD, and mRNA expressions of GST-pi, Cu, Zn-SOD increased remarkably in the clams exposed to combined pollutants. In this study, a significant interaction was observed for Cd and BaP accumulation in the clam and the current findings demonstrate the differences in antioxidant response of the biomarkers in clam to single contaminant and the mixtures.

Sex-related differences in rat liver microsomal enzymes and their induction by doxapram.

The effects of doxapram on the hepatic microsomal mono-oxygenase system of male and female rats were investigated. Male and female rats were administered doxapram (10-120 mg kg-1 day-1, i.p.) for 4 days. In female rats, administration of doxapram (20, 40, 60, 80, 100 and 120 mg kg-1) elevated the parameters in a dose-dependent manner while doxapram (100 and 120 mg kg-1) elevated the levels of cytochrome P450 and hexobarbitone hydroxylase in male rats. Doxapram (40 mg kg-1) caused induction of hepatic drug metabolism typified by an increase of hepatic microsomal cytochrome P450 content and activities of hexobarbitone hydroxylase, benzphetamine N-demethylase and ethylmorphine N-demethylase in female rats, but no change in male rats. These findings were supported by the results of SDS/polyacrylamide-gel electrophoresis. However, 7-ethoxycoumarin O-de-ethylase and arylhydrocarbon hydroxylase activities were significantly increased in male rats. NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities, and cytochrome b5 content were unaffected in rats of both sexes. The sex-dependent cytochrome P450 species may be selectively sensitive to the action of doxapram.

A Survey of Dietary Intake of Polychlorinated Dibenzo-p-dioxins, Polychlorinated Dibenzofurans, and Dioxin-like Coplanar Polychlorinated Biphenyls from Food During 2000–2002 in Osaka City, Japan

Studies of the dietary intake of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like coplanar polychlorinated biphenyls (dioxin-like PCBs), collectively referred as dioxins, were conducted using the total diet study (TDS) method with food purchased in Osaka City, Japan during 2000-2002. The daily intake of dioxin-TEQ (toxic equivalent) from food per adult person was estimated respectively as 104.24 pg TEQ/person/day in 2000, 72.73 pg TEQ/person/day in 2001, and 87.28 pg TEQ/person/day in 2002, corresponding to 2.08, 1.45, and 1.74 pg TEQ/kg body weight (bw)/day for an adult weighing 50 kg. The highest contribution ratio to the total intake of dioxin-TEQ was from fish and shellfish (group 10) in each year, accounting for 77-92%. The next highest contributor was meat and eggs (groups 11-A and 11-B). An annual decrease of the intake of dioxin-TEQ was not observed clearly. Otherwise, the dietary intake of non-2,3,7,8-substituted PCDDs/PCDFs were estimated together. The intake of 1,3,6,8-TeCDD came mostly from intake of fish and shellfish (group 10), green vegetable (group 7), and the other vegetables, including mushrooms and seaweed (group 8). In addition, the intake of 1,3,6,8-TeCDF, which reportedly antagonizes 2,3,7,8-TeCDD-mediated aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction activities, was an insufficient amount to suppress 2,3,7,8-TeCDD-mediated activities. Furthermore, we discussed different TEQ compositions of PCDDs/PCDFs to dioxin-like PCBs from food intake and in human samples and inferred that the difference was caused by low bioaccumulation properties of 3,3',4,4',5-PeCB (PCB 126).

Modulatory effect of Morus indica against two-stage skin carcinogenesis in Swiss albino mice: possible mechanism by inhibiting aryl hydrocarbon hydroxylase.

The modulatory effect of the methanolic extract of Morus indica on 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced oxidative stress and 7,12-dimethylbenz(a)anthracene induced and croton oil (0.5% per mouse/0.2 mL acetone, v/v) promoted skin tumourigenesis in Swiss albino mice was studied. The efficacy of the M. indica extract was also evaluated in-vitro by studying the inhibition of the activity and level of aryl hydrocarbon hydroxylase, cytochrome P450, DNA sugar damage in calf thymus DNA and Fe(++)/ascorbate-induced lipid peroxidation in microsomes of mice. Significant increases in the activity of antioxidant enzymes (P <0.001) and a concomitant decrease (P <0.001) in the cutaneous malondialdehyde level were observed at three doses of plant extract (2.5, 5.0 and 7.5 mg kg(-1)). Application of M. indica 1 h before each application of croton oil showed inhibitory effects on tumour promotion in terms of a reduction in the number of tumours/mouse and percentage of mice with tumours. It was also accompanied by an extension of the tumour latency period. TPA, which resulted in a rapid and transient stimulation of mouse epidermal ornithine decarboxylase activity (P <0.001), was inhibited dose dependently by pre-treatment with M. indica extract (P <0.001). The results suggest that M. indica extract may be useful as a therapeutic agent for cancer control as it blocks or suppresses events associated with chemical carcinogenesis.

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.

Potentiation of neurotoxicity of Lathyrus sativus by manganese: Alterations in blood–brain barrier permeability

Environmental factors have been speculated to play an important role in potentiating the neurotoxicity of Lathyrus sativus (LS). Hence, blood–brain barrier permeability and neurotoxicity studies were carried out in manganese- and LS-exposed animals. Dietary feeding of LS (80%) plus Mn (0.4 mg/100 g diet) for 90 days to guinea pigs showed significant (p < 0.05) decrease in brain nucleotidase and ATPase activities when compared to control or LS alone treated groups. Combined treatment of LS and Mn showed a significant (p < 0.05) decrease in neuronal aryl hydrocarbon hydroxylase (36–40%), ethoxyresorufin-O-deethylase (40–45%), glutathione-S-transferase (27–31%), and quinone reductase (24–25%) activities when compared to control and LS alone treated animals. Lipid peroxidation, a marker for membrane damage, was found to be relatively more enhanced (58–141%) along with significant (p < 0.05) depletion of GSH levels in LS+Mn-treated animals when compared to control, Mn alone, and LS alone treated groups. The neuronal catalase activity of lathyrus plus Mn-treated animals showed a pronounced decrease (37–49%) when compared to control, Mn, and lathyrus alone treated groups. On the contrary, glutathione peroxidase in brain of Mn and lathyrus alone treated animals indicated a respective increase (p < 0.05) of 18% and 20%, while the combined effect of lathyrus plus Mn exhibited an increase of almost 50% when compared to control guinea pigs. Single parenteral administration of Mn (15 mg/kg b.wt) to guinea pigs followed by single oral intubation of β-N-oxalyl-L-α, β-diamino propionic acid (ODAP, 75 mg/guinea pig) resulted in a significant increase (143%) in neuronal ODAP content. ODAP (50 mg/kg,iv) treatment to mice pretreated with MnCl2 (10 mg/kg b.wt for 3 days or 40 mg/kg b.wt for 1 day), caused an enhancement in blood–brain barrier (BBB) permeability (129–196%), while ODAP and Mn alone showed relatively less enhancement (66–87%). The lumbar region of LS+Mn showed a number of vacuolated areas of variegated size and chromatolytic neurons, along with a few degenerated neurons. These results suggest that Mn may potentiate the neurotoxicity of lathyrus/ODAP by altering the BBB permeability.

N-nitrosodimethylamine induced changes in the activities of carcinogen-metabolizing enzymes in the liver of male mice: role of glutathione and gossypol as antioxidants

The importance of nutrition in protecting living organisms from the toxic effects of environmental carcinogens has attracted the attention of many researchers. In order to exert their toxic and/or carcinogenic effects, most carcinogens need to be activated primarily by phase I drug-metabolizing enzymes including; cytochrome P450, cytochrome b5, arylhydrocarbon hydroxylase (AHH), N-nitroso-dimethylamine N-demethylase I (NDMA-N-dI), NADPH-cytochrome c reductase and the expression of cytochrome P450 2E1. Changes in the activity of these enzymes were determined in livers of male mice pretreated with either glutathione (GSH) or gossypol as antioxidants, for seven consecutive days before administration of NDMNA as single dose for two hours before decapitation of mice. The total hepatic content of cytochrome P450 and AHH activity were induced, whereas the activity of NDMA-N-dI and expression of cytochrome P450 2E1 were reduced after treatment of mice with NDMA as a single dose. On the other hand, preteatments of mice with either gossypol or GSH for seven consecutive days decreased the total cytochrome P450 content as well as AHH activity whereas the activity of NDMA-N-dI and the expression of cytochrome P450 2E1 increased. Treatment of mice with NDMA only induced the total hepatic content of cytochrome P450 and AHH activity. Interestingly pretreatment of mice with GSH for seven consecutive days before injection of NDMA was found to restore the induced cytochrome P450 and AHH activity caused by NDMA to their normal levels. However, pretreatment of mice with gossypol prior to administration of NDMA did not restore such activities to their normal levels. Treatment of mice with either GSH or gossypol induced the expression of cytochrome P450 2E1. Interestingly, gossypol/NDMA treated mice restored the induced cytochrome P450 2E1 expression, caused by gossypol, to its normal level. However, GSH/NDMA-treated mice did not restore the induced level of cytochrome P450 2E1 caused by GSH, to its normal level. It is concluded that, pretreatment of mice with GSH and gossypol prior to administration of NDMA induced the expression of cytochrome P450 2E1. Therefore, GSH and gossypol, could induce the toxicity and/or carcinogenicity of N-nitrosamines whereas they might protect the liver and probably other organs from the toxic and carcinogenic effects of other carcinogens such as polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene, through inhibition of total cytochrome P450 content and AHH activity. Key words: Aryl hydrocarbon hydroxylase, Cytochrome P450 2E1, N-nitrosodimethylamine N- demethylase, glutathione, antioxidants.

AHH activity, tissue dose and DNA damage in different tissues of the scallop Chlamys farreri exposed to benzo[ a]pyrene

A collaborative study was performed on scallops ( Chlamys farreri) exposed to 0.5, 3 and 10 μg/L benzo[ a]pyrene (B[ a]P) for 20 days. The levels of aryl hydrocarbon hydroxylase (AHH) activity, B[ a]P accumulation and DNA strand break were assayed in the gill and digestive gland. Results showed that AHH activity and B[ a]P accumulation were significantly related to B[ a]P dose. AHH activity was induced and then became stable gradually. B[ a]P accumulation increased first and showed an incoming plateau. DNA strand break levels in the 0.5 and 3 μg/L B[ a]P groups remained high and significantly different from control values until day 6, followed by a reduction in the gill and no recovery in the digestive gland. The 10 μg/L B[ a]P groups remained significantly lower than control until the end. These results suggested that the application of comprehensive indices may give information on the actual exposure of organisms to pollutants, and also information on toxic effects.

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).

CDNA cloning and characterization of feline CYP1A1 and CYP1A2

Deficiency of drug glucuronidation in the cat is one of the major reasons why this animal is highly sensitive to the side effects of drugs. The characterization of cytochrome P450 isoforms belonging to the CYP1A subfamily, which exhibit important drug oxidation activities such as activation of pro-carcinogens, was investigated. Two cDNAs, designated CYP1A-a and CYP1A-b, corresponding to the CYP1A subfamily were obtained from feline liver. CYP1A-a and CYP1A-b cDNAs comprise coding regions of 1554 bp and 1539 bp, and encode predicted amino acid sequences of 517 and 512 residues, respectively. These amino acid sequences contain a heme-binding cysteine and a conserved threonine. The cDNA identities, as well as the predicted amino acid sequences containing six substrate recognition sites, suggest that CYP1A-a and CYP1A-b correspond to CYP1A1 and CYP1A2, respectively. This was confirmed by the kinetic parameters of the arylhydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities of expressed CYPs in yeast AH22 cells and by the tissue distribution of each mRNA. However, theophylline 3-demethylation is believed to be catalyzed by CYP1A1 in cats, based on the high V max and low K m seen, in contrast to other animals. Because feline CYP1A2 had a higher K m for phenacetin O-deethylase activity with acetaminophen, which cannot be conjugated with glucuronic acid due to UDP-glucuronosyltransferase deficiency, it is supposed that the side effects of phenacetin as a result of toxic intermediates are severe and prolonged in cats.

Induction by topical corticosteroids of skin enzymes metabolizing carcinogenic hydrocarbons.

SUMMARY Epidermal aryl hydrocarbon hydroxylase activity of homogenates from mice treated for 7 days with various topical corticosteroid ointments was significantly higher than in preparations from animals receiving steroid-free ointment bases, or no treatment. As determined by 3,4-benzopyrene oxidation, enzyme activity was also greatly increased 24 h after small doses of various polycyclic hydrocarbons given by the intraperitoneal route. Inhibition of enzyme activity was observed in skin homogenates from both control and treated animals following addition in vitro of 7,8-benzoflavone, but not with dimethylsulphoxide.