Identification of a CYP3A form (CYP3A126) in fathead minnow ( Pimephales promelas ) and characterisation of putative CYP3A enzyme activity

Cytochrome P450-dependent monooxygenases (CYPs) are involved in the metabolic defence against xenobiotics. Human CYP3A enzymes metabolise about 50% of all pharmaceuticals in use today. Induction of CYPs and associated xenobiotic metabolism occurs also in fish and may serve as a useful tool for biomonitoring of environmental contamination. In this study we report on the cloning of a CYP3A family gene from fathead minnows (Pimephales promelas), which has been designated as CYP3A126 by the P450 nomenclature committee (GenBank no. EU332792). The cDNA was isolated, identified and characterised by extended inverse polymerase chain reaction (PCR), an alternative to the commonly used method of rapid amplification of cDNA ends. In a fathead minnow cell line we identified a full-length cDNA sequence (1,863 base pairs (bp)) consisting of a 1,536bp open reading frame encoding a 512 amino acid protein. Genomic analysis of the identified CYP3A isoenzyme revealed a DNA sequence consisting of 13 exons and 12 introns. CYP3A126 is also expressed in fathead minnow liver as demonstrated by reverse transcription PCR. Exposure of fathead minnow (FHM) cells with the CYP3A inducer rifampicin leads to dose-dependent increase in putative CYP3A enzyme activity. In contrast, inhibitory effects of diazepam treatment were observed on putative CYP3A enzyme activity and additionally on CYP3A126 mRNA expression. This indicates that CYP3A is active in FHM cells and that CYP3A126 is at least in part responsible for this CYP3A activity. Further investigations will show whether CYP3A126 is involved in the metabolism of environmental chemicals. Figure Induction of CYP3A activity by rifampicin and inhibition by diazepam in FHM cell

Mammalian epoxide hydrolases in xenobiotic metabolism and signalling

Epoxide hydrolases catalyse the hydrolysis of electrophilic—and therefore potentially genotoxic—epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)—an enzyme prone to detoxification—due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structur

Detoxification Strategy of Epoxide Hydrolase

The human microsomal epoxide hydrolase, a single enzyme, has to detoxify a broad range of structurally diverse, potentially genotoxic epoxides that are formed in the course of xenobiotic metabolism. The enzyme has developed a unique strategy to combine a broad substrate specificity with a high detoxification efficacy, by immediately trapping the reactive compounds as covalent intermediates and by being expressed at high levels for high trapping capacity. Computer simulation and experimental data as well as existing epidemiologic studies reveal this detoxification strategy as the mechanistic basis for a threshold in the tumorigenesis of mutagenic carcinogens

Chlordan

Chlordane [57-74-9] is used as an insecticide but is no longer approved in the European Union. The previous MAK value documentation and supplement do not reflect the current data situation of the substance. The MAK Commission decided that a new evaluation is not of high priority. The MAK value and the other classifications are therefore suspended and the substance is listed in the Section II c of the List of MAK and BAT Values for substances no longer evaluated

Aldrin

Aldrin [309-00-2] is used as an insecticide but is no longer approved in the European Union. The previous MAK value documentation and supplement do not reflect the current data situation of the substance. The MAK Commission decided that a new evaluation is not of high priority. The MAK value and the other classifications are therefore suspended and the substance is listed in the Section II c of the List of MAK and BAT Values for substances no longer evaluated

Sodium pyrithione

The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has re-evaluated sodium pyrithione [3811-73-2; 15922-78-8] considering all toxicological end points. Available publications and unpublished study reports are described in detail. Sodium pyrithione is neurotoxic in rats and rabbits, but not in monkeys. As there is no sufficient mechanistic explanation for the observed differences between the species, the rat as the most sensitive species is used for the derivation of a maximum concentration at the workplace (MAK value). The NOAEC in a 90-day inhalation study with rats is 1.1 mg/m3. In a chronic feeding study with rats, a NAEL of 0.16 mg/kg body weight and day is derived from the LOAEL of 0.5 mg/kg body weight and day. Both the NOAEC and the NAEL correspond to a MAK value of 0.2 mg/m3 for the inhalable fraction. As a systemic effect is critical, the substance remains classified in the Limitation Category II. As the initial half-life of sodium pyrithione is in the range of up to 2.8 hours, an excursion factor of 2 is assigned. In developmental toxicity studies, the most critical effects of sodium pyrithione are skeletal anomalies in rats. NOAELs for developmental effects are 2 mg/kg body weight and day after oral treatment of rats as well as 3 and 5 mg/kg body weight and day after dermal application to rats and rabbits, respectively. The differences between the NOAELs for rats and rabbits scaled to an inhalation concentration at the workplace and the MAK value are considered sufficient. Therefore, damage to the embryo or foetus is unlikely when the MAK value is not exceeded and sodium pyrithione is assigned to Pregnancy Risk Group C. Sodium pyrithione is still regarded as a non-genotoxic and non-carcinogenic substance. Skin contact may contribute significantly to systemic toxicity and sodium pyrithione remains designated with an “H” notation. Sensitization is not expected based on the limited data available

Triphenyl phosphate, isopropylated

he German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has evaluated triphenyl phosphate, isopropylated [68937-41-7] to derive a maximum concentration at the workplace (MAK value), considering all toxicity end points. Available unpublished study reports and publications are described in detail. Isopropylated triphenyl phosphate has no irritant effects on the skin of rats and rabbits, and is not, or at most minimally, irritating to the eyes of rabbits. It belongs to the group of organophosphates and shows the typical delayed organophosphate neurotoxicity (axonal degeneration). The neurotoxicity decreases with increasing isopropylation. However, the most sensitive toxicological end points following repeated exposures are histopathological changes in the adrenal gland and ovary. The LOAEC in 90-day inhalation studies in rats and in hamsters was 10 mg/m3, the lowest tested concentration. Oral studies according to OECD TG 422 and TG 408 have revealed a LOAEL of 25 mg/kg body weight and day in rats. Neurotoxicity tests in hens have yielded a NOAEL of 20 mg/kg body weight and day. After scaling these NOAELs to a concentration at the workplace, a MAK value of 1 mg/m3 is derived. As the systemic effect is critical, isopropylated triphenyl phosphate is assigned to Peak Limitation Category II with the default excursion factor of 2, as no specific toxicokinetic data are available. No developmental toxicity was observed at 260 mg isopropylated triphenyl phosphate/kg body weight and day. Therefore, no damage to the embryo or foetus has to be expected and isopropylated triphenyl phosphate is classified in Pregnancy Risk Group C. Isopropylated triphenyl phosphate is not genotoxic in vitro or in vivo nor does it have cell-transforming activity. No data on carcinogenicity are available. Overall, the available data do not indicate that the substance should be classified as a carcinogen or a germ cell mutagen. Sensitizing potential was not investigated with isopropylated triphenyl phosphate, and similar compounds have led to inconclusive results. Absorption through the skin is low and does not relevantly contribute to systemic toxicity

1,2-Dimethylhydrazine

The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) has re-evaluated the occupational exposure limit value (maximum concentration at the workplace, MAK value) of 1,2-dimethylhydrazine [540-73-8] considering all toxicological end points. Relevant studies were identified from a literature search. Chronic and subchronic exposure induced adverse effects on the liver, heart and kidneys of mice and the liver and bile ducts of mini-pigs and guinea pigs. In dogs, 1,2-dimethylhydrazine caused adverse effects on the liver. The critical effect of 1,2-dimethylhydrazine is its carcinogenic potential. In carcinogenicity studies, 1,2-dimethylhydrazine induced various intestinal and vascular tumours such as haemangiosarcomas in addition to lung tumours in rodents after oral and intraperitoneal application. Particularly noteworthy is the high incidence of colon carcinomas in rats, which was observed both after acute and chronic application. Additionally, tumours of the digestive system were caused in hamsters and monkeys after subcutaneous or intramuscular injection. On the basis of the carcinogenic effects induced in several animal species, 1,2-dimethylhydrazine remains classified in Carcinogen Category 2. It is not possible to derive a MAK value. Enzymatic activation of 1,2-dimethylhydrazine leads to highly reactive metabolites, such as the well-known procarcinogen methylazoxymethanol, which are able to methylate DNA. The substance is clastogenic and mutagenic in somatic cells in vitro and in vivo. Additionally, in mouse testes, 1,2-dimethylhydrazine was found to inhibit DNA synthesis after oral application and to methylate DNA after subcutaneous application. Therefore, the substance has been classified in Germ Cell Mutagenicity Category 3 A. Although no valid studies considering the dermal absorption of 1,2-dimethylhydrazine are available, the “H” designation has been retained because of the low dermal LD50 values in animals and the potential for genotoxic effects after dermal application. Although no studies considering the sensitizing potential of 1,2-dimethylhydrazine are available, the “Sh” designation has been retained because of its structural similarity with the known contact allergen hydrazine

Antimony and its inorganic compounds except for stibine

The German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) summarized and re-evaluated the data on the genotoxicity and carcinogenicity of antimony [7440-36-0] and its inorganic compounds except for stibine. The critical effects of antimony and its inorganic com- pounds are the carcinogenic effects on the lung after inhalation exposure in rats and mice; similar effects may be induced in humans. Overall, the available epidemiological studies indicate that antimony and its inorganic compounds have carcinogenic effects on the human lung. However, because the persons examined were exposed to mixtures of substances and no data for concentrations are available, the classification cannot be made on the basis of these studies alone. A recent 2-year carcinogenicity study in male and female rats and mice showed that exposure to antimony trioxide particles causes lung neoplasms. Mice reacted more sensitively than rats, developing neoplastic lesions beginning at the lowest antimony trioxide concentration of 3 mg/m3 (2.5 mg Sb/ m3). As a NOAEC for lung tumours and lung effects cannot be derived from the ani- mal or human data and a NOAEC cannot be determined for possible mechanisms of action, no maximum concentration at the workplace (MAK value) can be established, thereby confirming the classification of antimony and its inorganic compounds in Carcinogen Category 2. The clastogenicity of inorganic antimony compounds in vitro is well established. In a recent study, exposure to antimony trioxide by inhalation for 12 months increased the number of micronucleated erythrocytes and DNA strand breaks in lung cells in male and female mice, but not in male or female rats. The lowest effective concentration was 3 mg/m3 and led to increased DNA strand breaks in lung tissue in male mice. Thus, trivalent antimony was shown to induce genotoxic effects in soma cells after inhalation. This, together with evidence that the substance reaches the testes and ovaries, led to the classification of antimony and its inorganic compounds in Germ Cell Mutagenicity Category 3 A. There are still no reliable positive data for sensitizing effects in humans and no positive results from animal experiments or in vitro investigations. Therefore, antimony and its inorganic compounds continue not to be designated with the “Sh” or “Sa” notation

Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,S′]dioxodi-µ-thioxodimolybdenum

The German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) summarized and evaluated the data for bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,Sʹ]dioxodi-μ-thioxodimolybdenum [68958-92-9; 72030-25-2] to derive an occupational exposure limit value (maximum concentration at the workplace, MAK value) considering all toxicological end points. The documentation is based primarily on the REACH registration dossier. A study described in the REACH registration dossier that was carried out according to OECD Test Guideline 422 found increased kidney weights in male rats and elevated levels of thyroid-stimulating hormone in female rats at 100 mg/kg body weight and day. As the percentage change in kidney weights was not specified and the study was not made available to the Commission, a maximum concentration at the workplace (MAK value) cannot be derived and bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,Sʹ]dioxodi-μ-thioxodimolybdenum has been assigned to Section II b of the List of MAK and BAT Values. Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,Sʹ]dioxodi-μ-thioxodimolybdenum was not found to be genotoxic in vitro; neither in vivo genotoxicity data nor carcinogenicity studies are available. There is no clear evidence of a contact sensitizing potential and no data for sensitization of the respiratory tract. Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,Sʹ]dioxodi-μ-thioxodimolybdenum is not expected to be taken up via the skin in toxicologically relevant amounts