Up-regulation of the alligator CYP3A77 gene by toxaphene and dexamethasone and its short term effect on plasma testosterone concentrations.
In this study we describe an alligator hepatic CYP3A gene, CYP3A77, which is inducible by dexamethasone and toxaphene. CYP3A plays a broad role in biotransforming both exogenous compounds and endogenous hormones such as testosterone and estradiol. Alligators collected from sites in Florida that are contaminated with organochlorine compounds exhibit differences in sex steroid concentrations. Many organochlorine compounds induce CYP3A expression in other vertebrates; hence, CYP3A induction by organochlorine contaminants could increase biotransformation and clearance of sex steroids by CYP3A and provide a plausible mechanism for the lowering of endogenous sex steroid concentrations in alligator plasma. We used real time PCR to examine whether known and suspected CYP3A inducers (dexamethasone, metyrapone, rifampicin, and toxaphene) up-regulate steady state levels of hepatic CYP3A77 transcript to determine if induction patterns in female juvenile alligators are similar to those reported in other vertebrates and whether toxaphene, an organochlorine compound found in high concentrations in Lake Apopka alligators, induces this gene. Estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), and steroid-xenobiotic receptor (SXR) transcripts were also measured to determine whether any of these nuclear receptors are also regulated by these compounds in alligators. Dexamethasone (4.2-fold) and toxaphene (3.5-fold) significantly induced CYP3A77 gene transcript, whereas rifampicin (2.8-fold) and metyrapone (2.1-fold) up-regulated ERbeta after 24h. None of the compounds significantly up-regulated AR, ERalpha, GR, PR, or SXR over this time period. Plasma testosterone (T) did not change significantly after 24h in alligators from any of the treatment groups. Dexamethasone treated animals exhibited a strong relationship between the 24h plasma T concentrations and CYP3A77 (R(2)=0.9, positive) and SXR (R(2)=0.77, negative) transcripts, which suggests that the expression of these genes is related to plasma T in alligators. In light of our findings, we hypothesized that higher steady state CYP3A77 (and possibly SXR) gene expression would be observed in alligators collected from Lake Apopka, a polluted lake containing organochlorine compounds known to induce CYP3A isoforms in other taxa. Therefore, we measured basal levels of CYP3A77 and SXR gene transcripts in wild juvenile alligators collected from Orange Lake (reference lake), Lake Woodruff (reference lake), and Lake Apopka (contaminated lake). We found that no differences existed in CYP3A77 or SXR gene expression among animals from the lakes sampled suggesting that exposure to organochlorine compounds at concentrations present in Lake Apopka does not lead to variation in the expression of these genes, although capture stress could be interfering with these results since the glucocorticoid dexamethasone induces CYP3A77 transcript in alligators.
Gunderson
M P
MP
Department of Zoology, University of Florida, P.O. Box 118525, Gainesville, FL 32611-8525, United States. mgunders@uvic.ca
Kohno
S
S
Blumberg
B
B
Iguchi
T
T
Guillette
L J
LJ
D000489:Alligators and Crocodiles / Q000097:blood / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D003907:Dexamethasone / Q000633:toxicity*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005938:Glucocorticoids / Q000633:toxicity; D007306:Insecticides / Q000633:toxicity; D008297:Male; D008797:Metyrapone / Q000633:toxicity; D008969:Molecular Sequence Data; D000077297:Pregnane X Receptor; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000235:genetics; D011987:Receptors, Steroid / Q000096:biosynthesis / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012293:Rifampin / Q000633:toxicity; D013739:Testosterone / Q000097:blood*; D014112:Toxaphene / Q000633:toxicity*; D015854:Up-Regulation / Q000187:drug effects; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D005938:Glucocorticoids; D007306:Insecticides; D000077297:Pregnane X Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014874:Water Pollutants, Chemical; D013739:Testosterone; D003907:Dexamethasone; D014112:Toxaphene; D051544:Cytochrome P-450 CYP3A; D012293:Rifampin; D008797:Metyrapone