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The interaction of the organotin fungicide triphenyltin chloride (TPT) with fish microsomal monooxygenase systems has been studied in vitro and in vivo in the marine fish scup (Stenotomus chrysops). In vitro incubation of fish liver microsomes with TPT resulted in the conversion of about 40% of the native total spectral P450 to P420. In addition, a strong concentration-related inhibition of ethoxyresorufin O-deethylase (EROD) activity was observed, with a complete loss at 1.0 mM TPT. Pentoxyresorufin-O-dealkylase (PROD) activity was inhibited only at the highest concentration tested. This suggests either some specificity for the EROD catalyst CYP1A1, or a loss of reductant NADPH cytochrome c reductase as the cause. Further in vitro incubations showed that NADPH, but not NADH, cytochrome c reductase was strongly inhibited at 100 microM TPT and higher. To further investigate this effect, fish were injected with single doses of 5, 25 and 50 microM TPT (1.9, 9.6 and 19.3 mg kg-1 TPT), and 24 and 48 h later, hepatic microsomes were analyzed for total P450 content, EROD activity, NAD(P)H cytochrome c reductase, and the content of three CYP forms. EROD activity tended to be decreased in TPT-treated scup, with the response being stronger after 48 than 24 h. No significant conversion of spectrally determined P450 to cytochrome P420 was found, and cytochrome b5 was not affected. However, both NAD(P)H cytochrome c reductases were significantly inhibited at all concentrations. Immunoblot analysis showed reduction of CYP1A1 content at all doses, being significant at 25 mM after 48 h, but no decrease in CYP3A-like protein, the dominant catalyst of testosterone 6 beta-hydroxylation, nor CYP2B-like protein, the major contributor to indicates significant effects of TPT at high concentrations on fish hepatic CYP1A1 protein, EROD activity and the reductases. TPT seems to act more specifically on CYP1A1 than on other CYP forms. These findings combined with those of our previous studies (Brüschweiler BJ, Würgler FE, Fent K. Environ Toxicol Chem 1996;15:827-735; Fent K, Bucheli TD. Aquat Toxicol 1994;28:107-126; Fent K, Stegeman JJ. Aquat Toxicol 1991;20:159-168; Fent K, Stegeman JJ. Aquat Toxicol 1993;24:219-240) indicate a general degenerative effect of organotins on the fish microsomal monooxygenase system, although some differences are seen between the organotins, and between species. We conclude that these effects of organotins have consequences for use of CYP1A as a biomarker and endocrine disruption.

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that controls the expression of cytochrome P450 1A (CYP1A) genes in response to halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand and normal physiologic function of this protein are as yet unknown. One approach to understanding AHR function and significance is to determine the evolutionary history of this receptor and of processes such as CYP1A induction that are controlled by the AHR in mammals. In these studies, AHR function was evaluated in representative cartilaginous fish (little skate, Raja erinacea) and jawless fish (sea lamprey, Petromyzon marinus and Atlantic hagfish, Myxine glutinosa), using CYP1A induction as a model AHR-dependent response. Treatment of skate with beta-naphthoflavone (BNF) caused an 8-fold increase in hepatic ethoxyresorufin O-deethylase (EROD) activity as well as a 37-fold increase in the content of immunodetectable CYP1A protein. Evidence of CYP1A inducibility was also obtained for another cartilaginous fish, the smooth dogfish Mustelus canis. In contrast, hepatic EROD activity was not detected in untreated lamprey nor in lamprey treated with 3,3'4,4'-tetrachlorobiphenyl (TCB), a potent AHR agonist in teleosts. A possible CYP1A homolog was detected in lamprey hepatic microsomes by one of three antibodies to teleost CYP1A, but expression of this protein was not altered by TCB treatment. CYP1A protein and catalytic activity were measurable in hagfish, but neither was induced after treatment with TCB. These results suggest that the AHR-CYP1A signal transduction pathway is highly conserved in gnathostomes, but that there may be fundamental differences in AHR signaling or AHR-CYP1A coupling in agnathan fish. Agnathan fish such as hagfish and lamprey may be interesting model species for examining possible ancestral AHR functions not related to CYP1A regulation.

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23-2.1 nmol/min/mg and 0.5-11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with beta-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6 beta- and 16 beta-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.