CYP3C gene regulation by the aryl hydrocarbon and estrogen receptors in zebrafish.

Cytochrome P450 (CYPs) enzymes are critical for the metabolism of exogenous and endogenous compounds. In mammals, the CYP3s are arguably the most important xenobiotic metabolizing enzymes and are all contained within the CYP3A subfamily. In fish, CYP3s include CYP3A and multiple subfamilies unique to the teleost lineage. The goal of this study was to provide insight on the regulation of genes in the CYP3C subfamily. Zebrafish, which have 4 CYP3C genes, were exposed to 17ß-estradiol (E2; 0.001-10 µM) or ß-naphthoflavone (ßNF; 0.005-1 µM), prototypical ligands of the estrogen receptor (ER) and the aryl hydrocarbon receptor (AhR), respectively. Gene expression was measured in the liver, intestine and gonads using quantitative PCR. CYP1A and vitellogenin (VTG) gene expression were used as positive controls for AhR and ER regulation, respectively. Exposure to ßNF resulted in the dose-dependant induction of CYP1A and CYP3C genes in the female intestine but not in the liver. E2 exposure resulted in the induction of all CYP3Cs in the male intestine and in the female liver. VTG was induced in both female and male livers. CYP3C3 and CYP3C4 were induced in the testis; CYP3C1 and CYP3C4 were slightly induced in the ovary. The time-course of gene induction was investigated in the liver and intestine after exposure to ßNF (0.5 µM) and E2 (0.1 µM). Inducible genes were up-regulated within 12 h after exposure. These data support a role for the AhR and ER in the regulation of CYP3Cs. Overall, the induction of CYP3Cs by AhR and ER ligands is different from mammalian CYP3A and may suggest a functional role for CYP3Cs that involves planar aromatic hydrocarbons and steroids.

Expression patterns of cytochrome P450 3B and 3C genes in model fish species.

Cytochrome P450 (CYP) 3 enzymes are highly expressed in detoxification organs and play an important role in xenobiotic metabolism. In fish, the CYP3 family is diversified and includes several subfamilies (CYP3B, CYP3C, and CYP3D) not found in mammals. The functional role and expression patterns of these novel genes are unknown. In this study, the expression patterns of novel teleost CYP3 genes were determined in medaka(Oryzias latipes; CYP3B4, CYP3B5, CYP3B6) and zebrafish (Danio rerio; CYP3C1, CYP3C2, CYP3C3, CYP3C4), two important model fish species. Expression was quantified with real time PCR in multiple internal organs from adult male and female fish. CYP3C gene expression was determined in zebrafish embryos. Expression in all organs was detected for all genes, except for CYP3B4 in male organs. CYP3C1, CYP3C3, CYP3B4, CYP3B5, and CYP3B6 were more highly expressed in liver and/or intestine from at least one gender, suggesting a role in xenobiotic metabolism. Expression of CYP3C1 and CYP3B5 in olfactory rosette was comparable to liver. CYP3C1, CYP3C4, CYP3B5 and CYP3B6 expression was higher in the female organs; CYP3C2 and CYP3B5 were higher in testis. Estrogen and androgen response elements were found upstream of the start site of many of these genes raising the hypothesis that they are under steroid regulation. CYP3C1-3 were expressed in all developmental stages examined and appear to be maternally deposited. The expression patterns suggest that some of these CYP genes are involved in xenobiotic metabolism.

Effects of chronic, parental pharmaceutical exposure on zebrafish (Danio rerio) offspring.

In this study we explored how parental exposure to pharmaceuticals influences reproduction in offspring. Adult zebrafish (Danio rerio) were exposed for 6 weeks to 10 µgL(-1) of carbamazepine (CBZ) and gemfibrozil (GEM), two commonly prescribed drugs. Embryos were collected, reared in clean water until sexual maturity and then assessed for reproductive output, courtship, sperm function and organ histology. While 34% of the control pairs produced clutches, only 11% of the fish with CBZ exposed parents or 17% of the fish with GEM exposed parents produced clutches. Reciprocal crosses indicated that exposure in males had more profound reproductive effects. When a control F1 male was crossed with either a F1 female whose parents were CBZ or GEM exposed; no differences were observed in embryo production compared to controls. However, when a control F1 female was crossed with either a CBZ or GEM F1 male, 50% less embryos were produced. Male courtship was reduced in both CBZ and GEM F1 fish but the deficits in courtship displays were drug specific. Compared to control males, the sperm from GEM F1 males had shorter head lengths and midpieces whereas sperm from CBZ F1 males had longer midpieces. Although it remains unclear how specifically these morphological differences influenced sperm velocity, the sperm from GEM F1 males and from CBZ F1 males swam faster than the sperm of control F1 at 20s post activation. No significant differences were observed in the histology of the liver, kidney and gonads across treatment groups. These data are important as they show that chronic, low dose pharmaceutical exposure of parental fish is sufficient to cause significant reproductive effects in offspring.