The human carboxylesterase 1 (CES1) gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. During the course of a study of the pharmacokinetics of the methyl ester racemic psychostimulant methylphenidate, profoundly elevated methylphenidate plasma concentrations, unprecedented distortions in isomer disposition, and increases in hemodynamic measures were observed in a subject of European descent. These observations led to a focused study of the subject's CES1 gene. DNA sequencing detected two coding region single-nucleotide mutations located in exons 4 and 6. The mutation in exon 4 is located in codon 143 and leads to a nonconservative substitution, p.Gly143Glu. A deletion in exon 6 at codon 260 results in a frameshift mutation, p.Asp260fs, altering residues 260-299 before truncating at a premature stop codon. The minor allele frequency of p.Gly143Glu was determined to be 3.7%, 4.3%, 2.0%, and 0% in white, black, Hispanic, and Asian populations, respectively. Of 925 individual DNA samples examined, none carried the p.Asp260fs, indicating it is an extremely rare mutation. In vitro functional studies demonstrated the catalytic functions of both p.Gly143Glu and p.Asp260fs are substantially impaired, resulting in a complete loss of hydrolytic activity toward methylphenidate. When a more sensitive esterase substrate, p-nitrophenyl acetate was utilized, only 21.4% and 0.6% catalytic efficiency (V(max)/K(m)) were determined in p.Gly143Glu and p.Asp260fs, respectively, compared to the wild-type enzyme. These findings indicate that specific CES1 gene variants can lead to clinically significant alterations in pharmacokinetics and drug response of carboxylesterase 1 substrates.

The Leydig cell of the testis is the only cell in the male that has the capacity to synthesize testosterone from cholesterol. Testosterone is critical during fetal development for male sexual differentiation, and postnatally for initiation and maintenance of spermatogenesis and the expression of the male secondary sex characteristics. The biosynthesis of testosterone requires the activities of four enzymes, cholesterol side-chain cleavage enzyme (P450scc), 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD), 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)), and 17-ketosteroid reductase. The expression of these enzymes appears to be regulated by different mechanisms. The recent isolation of the mouse cDNAs and structural genes that encode these enzymes has enabled us to begin to investigate the regulation of their expression at the molecular level. This review discusses the regulation by cAMP and steroids of three enzymes in Leydig cells, P450scc, P450(17 alpha), and 3 beta HSD, as well as characterization of the promoters of the mouse genes that encode P450scc and P450(17 alpha).

The QuikChange Multi Site-Directed Mutagenesis Kit is a simple and efficient method for introducing point mutations at up to five sites simultaneously in plasmid DNA templates. Here we used the QuikChange Multi kit with degenerate (one codon) primers to introduce all possible amino acids at selected sites in the lacZ gene. In reactions employing two or three degenerate primers, diverse libraries (10(4)-10(5) mutants/reaction) are created consisting of random combinations of mutations at two or three different sites. This method provides a one-day procedure for performing site-directed saturation mutagenesis and, when coupled with a suitable screening assay, should greatly facilitate the process of evaluating alternative amino acid chain substitutions at key residues and evolving protein function.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Carcinogenesis 19, 1623-1629; Lewis et al., 2001, TOXICOL: Sci. 62, 46-53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 microg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring.

An uncharacterized murine cDNA clone was identified and, through sequence, phylogenetic, and functional analysis, determined to encode the newest member of the organic anion transporter family, organic anion transporter 5 (Oat5; Slc22a19). The Oat5 cDNA clone contained an insert 1,964 bp in length with a predicted open reading frame (from bp 84 to bp 1,739) coding for a peptide 551 amino acids long. Slc22a19 was localized to mouse chromosome 19 near the genes encoding Oat1 ( Slc22a6) and Oat3 ( Slc22a8). Northern blot analysis revealed Oat5 is highly expressed in the kidney of adult mice and rats. No sexual dimorphism in renal or hepatic expression of Oat5 was observed. Unlike Oat1–3, Oat5 expression was not detected in the choroid plexus of either mice or rats. Murine Oat5-expressing Xenopus laevis oocytes supported increased accumulation of the mycotoxin ochratoxin A, compared with water-injected control oocytes. This uptake was significantly inhibited by probenecid and the organic anions 2,4-dichlorophenoxyacetic acid, salicylate, and estrone sulfate but not by para-aminohippurate or urate. Transport of ochratoxin A by murine Oat5 was saturable, with an estimated Km of 2.0 ± 0.45 μM. Oat5-mediated transport was neither cis-inhibited nor trans-stimulated by the dicarboxylate glutarate. Uptake was also completely unaffected by short-circuiting of the membrane potential. Thus the motive forces behind Oat5 function, which provide insight into its membrane localization, need to be further resolved. These data demonstrate for the first time that this newly identified gene encodes a protein that functions as an organic anion transporter.