Organochlorine contaminants in loggerhead sea turtle blood: extraction techniques and distribution among plasma and red blood cells.

Few studies have described the organochlorine (OC) contaminant concentrations found in sea turtle tissues. These studies have relied on the opportunistic sampling of either eggs or tissues from stranded carcasses. In this study, the use of whole blood samples as well as both blood components (plasma and red blood cells) were examined as a non-destructive alternative for monitoring OCs in free-ranging loggerhead sea turtles (Caretta caretta). Blood samples were collected from juvenile loggerhead sea turtles (n = 12) captured in Core Sound, North Carolina, USA and analyzed for 55 polychlorinated biphenyl (PCB) congeners and 24 OC pesticides by gas chromatography with electron capture detection and mass spectrometry. Using pooled loggerhead sea turtle whole blood, three different liquid:liquid extraction techniques were compared. Results were similar in terms of recovery of internal standards, lipids, and OC concentrations. An extraction technique, employing formic acid and 1:1 methyl-tert-butyl-ether: hexane, was found to be satisfactory. This method was applied to the extraction of OCs from whole blood, plasma, and red blood cell (RBC) samples from five loggerhead sea turtles. Plasma contained the highest OC concentrations on a wet mass basis, followed by whole blood and RBCs. The majority of each OC compound was found in the plasma rather than the RBCs, suggesting that OC compounds preferentially partition into the plasma. On average (SD), 89.4% (3.1 %) of total PCBs, 83.4% (11.9%) of total chlordanes, 74.3% (15.1%) of mirex, 72.6% (4.8%) of total DDTs, and 80.1% (16.6%) of dieldrin were found in the plasma. The concentrations of total PCBs, mirex, total chlordanes, and total DDTs measured in both components of the blood significantly correlated to those in whole blood. These are the first reported OC concentrations in sea turtle blood. They were found to be similar to previously reported levels in blood components of humans and of reptiles from relatively clean sites, but lower than those measured in blood of fish-eating birds and marine mammals. The results indicate that blood, preferably plasma, can be used to detect and monitor OC contaminants in loggerhead sea turtles.

Characterization of a cDNA for the unexpressed form of cytochrome P-450g from the (-g) rat and differentiation of its mRNA from that of the (+g) phenotype using specific oligoprobes.

Our laboratory recently isolated a cDNA for cytochrome P-450g (IIC13), a male-specific, highly polymorphic P-450 isozyme, from livers of the high phenotype (+g) of Sprague-Dawley rats [McClellan-Green et al. (1989) Biochemistry 28, 5832-5839]. Hybridization studies using a specific oligonucleotide probe for P-450 (+g) indicated that equivalent amounts of P-450g mRNA were present in livers of both the high and low phenotypes (+g and -g) of male Sprague-Dawley, Fischer (inbred -g), or ACI (inbred +g) rats. In the present study, we isolated one full-length and one nearly full-length cDNA clone coding for the unexpressed form of cytochrome P-450g from a cDNA library constructed from mRNA from a (-g) male Sprague-Dawley rat. The longest cDNA had an open reading frame of 1473 nucleotides which coded for a 490 amino acid polypeptide of Mr 55,839. Although the 5'-noncoding leader sequence and the 3'-noncoding region were unchanged, the coding sequence of the (-g) phenotype differed from that of the cDNA isolated from the (+g) phenotype by nine bases changes. These base changes would result in seven amino acid differences between the protein sequences for the two phenotypes. Two specific oligonucleotide probes for (+) P-450g and (-) P-450g containing three base differences between the (+g) and (-g) sequences hybridized differentially to mRNA from the (+g) and (-g) phenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of male-specific rat hepatic cytochrome P-450g (P-450IIC13) by androgens and the pituitary.

The present study examines the hormonal regulation male-specific cytochrome P-450g (IIC13) and its mRNA. Neonatal gonadectomy of male rats abolished hepatic expression of P-450g and its mRNA in adulthood, while ovariectomy had little effect. Neonatal administration of testosterone to neonatally gonadectomized male or female rats partially masculinized expression of P-450g and its mRNA, and postpubertal administration of testosterone (testosterone capsules implanted at 5 weeks) completely masculinized their expression. However, castration of male rats at puberty (5 weeks) had no effect on P-450g or its mRNA at 10 weeks. Male-specific development of P-450g and P-450 M-1 (IIC11) mRNA were imprinted similarly by testosterone. However, hypophysectomy experiments demonstrated that the two male-specific forms of P-450 are regulated quite differently. Hypophysectomy of male rats decreased hepatic content of P-450 M-1 mRNA by approximately 50%, and intermittent injections of growth hormone completely restored this mRNA. In contrast, hypophysectomy of male rats increased P-450g and its mRNA by approximately 50%, while intermittent injections of growth hormone produced a slight decrease. Hypophysectomy of female rats increased P-450g and its mRNA to adult male levels, but produced only a small increase in P-450 M-1 mRNA. Continuous infusion of growth hormone into sham hypophysectomized male rats (to mimic the female growth hormone pattern) resulted in a complete loss of P-450g and its mRNA. These results indicate that the expression of P-450g is not dependent on the male pulsatile growth hormone pattern, but suggest instead that the continuous secretion of growth hormone suppresses P-450g in the female rat.

Characterization of a cDNA for rat P-450g, a highly polymorphic, male-specific cytochrome in the P-450IIC subfamily.

Cytochrome P-450g (IIC13) is a highly polymorphic, male-specific rat liver isozyme which is a member of the P-450IIC subfamily. A cDNA, c5126 (1737 bp), for P-450g was isolated from a lambda gt11 library synthesized from (+g) male rat liver mRNA. Sequence analysis of the clone, c5126, revealed an open reading frame of 1473 nucleotides, which encodes for a 490 amino acid polypeptide possessing the 30 NH2-terminal residues reported for cytochrome P-450 (M-3) (P-450g) [Matsumoto et al. (1986) J. Biochem. 100, 1359-1371]. A high degree of sequence similarity (greater than 70%) exists between c5126 and the published sequences of cDNAs for members of the IIC subfamily, while its sequence similarity to other subfamilies (IA, IIB, and IIIA) was much lower (less than 55%). RNA blot analysis utilizing an oligonucleotide probe specific for P-450g revealed that P-450g mRNA was expressed in livers of male but not female Sprague-Dawley (CD) and ACI rats, indicating that the sex difference was regulated pretranslationally. Furthermore, expression of P-450g mRNA was age dependent in livers of male ACI rats (a homozygous, phenotypically high P-450g strain). However, the mRNA for P-450g was expressed equally in livers of outbred male CD rats representing either the high (+g) or the low (-g) phenotype and of inbred ACI rats (+g) representing the high phenotype, indicating that the defect in (-g) rats does not reflect differences in expression of P-450g mRNA.