Seasonal expression of P450c17 and 5α-reductase-2 in the scented gland of male muskrats (Ondatra zibethicus).

Cytochrome P450 17A1 (P450c17) is the key enzyme required for the production of androgenic sex steroids by converting progestogens to androgens. 5α-reductases are enzymes that convert testosterone (T) to dihydrotestosterone (DHT), which has a greater affinity for androgen receptors (AR) and stronger action than T. Our previous studies revealed that the scented glands of male muskrats expressed AR during the breeding and nonbreeding seasons. To further seek evidence of the activities of androgens in scented glands, the expression patterns of P450c17 and 5α-reductase 2 were investigated in the scented glands of male muskrats during the breeding and nonbreeding seasons. The weight and size of scented glands in the breeding season were significantly higher than those of the nonbreeding season. Immunohistochemical data showed that P450c17 and 5α-reductase 2 were presented in the glandular cells and epithelial cells of scented glands in both the seasons. The protein and mRNA expression of P450c17 and 5α-reductase 2 were significantly higher in the scented gland during the breeding season than those during the nonbreeding season. In addition, the levels of DHT and T in the scented gland were remarkably higher during the breeding season. Taken together, these results suggested that the scented glands of male muskrats were capable of locally synthesizing T and DHT, and T and DHT might play an important role in the scented glandular function via an autocrine or paracrine manner.

CYP341B14: a cytochrome P450 involved in the specific epoxidation of pheromone precursors in the fall webworm Hyphantria cunea.

Two of the four sex pheromone components in the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene, possess an epoxy ring within their molecules. These compounds have been suggested to be biosynthesized from dietary linolenic acid via the following enzymatic reactions; chain elongation, terminal desaturation (in the case of the latter component), decarboxylation, and epoxidation. The last step of this biosynthesis, epoxidation, is known to occur specifically in the sex pheromone gland of females. We identified the enzyme involved in the epoxidation of pheromone precursors by focusing on cytochromes P450, which are known to catalyze the oxidation of various compounds. Three P450-like sequences (Hc_epo1, Hc_epo2, and Hc_epo3) were identified in the cDNA library prepared from the sex pheromone gland of H. cunea. Among these clones, only Hc_epo1 was specifically expressed in the pheromone gland. The full-length sequence of Hc_epo1 contained an ORF of 1527 bp, which encoded a protein of 509 amino acids with a predicted molecular weight of 57.9 kDa. The deduced Hc_epo1 amino acid sequence possessed the characteristics of P450. A phylogenetic analysis of the sequence indicated that Hc_epo1 belonged to the CYP341B clade in the CYP341 family. Therefore, it was named CYP341B14. A subsequent functional assay using Sf-9 cells transiently expressing CYP341B14 demonstrated that this P450 protein was able to specifically epoxidize a (Z)-double bond at the 9th position in the pheromone precursor, (3Z,6Z,9Z)-3,6,9-henicosatriene.

Oral benzo[a]pyrene: understanding pharmacokinetics, detoxication, and consequences--Cyp1 knockout mouse lines as a paradigm.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH); this ubiquitous environmental carcinogenic agent is found in tobacco smoke, charcoal-grilled foods, and PAH-contaminated surfaces of roofs, playgrounds, and highways. Cytochrome P450 1 wild-type, Cyp1a2(-/-), Cyp1b1(-/-), or Cyp1a2/1b1(-/-) knockouts, and mice with Cyp1a1 expression deleted in hepatocytes can ingest large oral BaP doses (125 mg/kg/d) without apparent toxicity. Cyp1a1(-/-) and Cyp1a1/1a2(-/-) knockouts and mice with Cyp1a1 expression deleted in gastrointestinal (GI) tract epithelial cells develop immunotoxicity and die within 32 days, indicating that GI tract inducible CYP1A1 is absolutely required for detoxication of oral BaP. Cyp1a1/1b1(-/-) and Cyp1a1/1a2/1b1(-/-) mice are rescued from immunosuppression and early death due to absent metabolic activation of BaP by CYP1B1 in immune cells. Ten-fold lower oral BaP doses result in adenocarcinoma of the proximal small intestine (PSI) in Cyp1a1(-/-) mice; Cyp1a1/1b1(-/-) double-knockout mice show no PSI cancer but develop squamous cell carcinoma of the preputial gland duct (PGD). BaP-metabolizing CYP1B1 in the PSI and CYP3A59 in the PGD are the most likely candidates to participate in tumor initiation in the epithelial cells of these two tissues; oncogenes and tumor-suppressor genes upregulated and downregulated during tumorigenesis are completely different between these tissues. This "oral BaP Cyp1" mouse paradigm represents a powerful teaching tool, showing that gene-environment interactions depend on route-of-administration: the same oral, but not intraperitoneal, BaP exposure leads to dramatic differences in target-organ toxicity and tumor type as a function of dose and Cyp1 genotype.

Biosynthetic pathways of the sex pheromone components and substrate selectivity of the oxidation enzymes working in pheromone glands of the fall webworm, Hyphantria cunea.

The fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae), is a harmful polyphagous defoliator. Female moths produce the following four pheromone components in a ratio of about 5:4:10:2; (9Z,12Z)-9,12-octadecadienal (I), (9Z,12Z,15Z)-9,12,15-octadecatrienal (II), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene (III), and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene (IV). Although ¹³C-labeled linolenic acid was not converted into trienal II at the pheromone glands of H. cunea females, GC-MS analysis of an extract of the pheromone gland treated topically with ¹³C-labeled linolenyl alcohol showed the aldehyde incorporating the isotope. Other C18 and C19 fatty alcohols were also oxidized to the corresponding aldehydes in the pheromone gland, indicating a biosynthetic pathway of IIvia linolenyl alcohol and low substrate selectivity of the alcohol oxidase in the pheromone gland. On the other hand, epoxydiene III was expected to be produced by specific 9,10-epoxidation of the corresponding C21 trienyl hydrocarbon, which might be biosynthesized from dietary linolenic acid in oenocytes and transported to the pheromone gland. The final biosynthetic step in the pheromone gland was confirmed by an experiment using deuterated C21 triene, which was synthesized by the chain elongation of linolenic acid and LiAlD4 reduction as key reactions. When the labeled triene was administered to the female by topical application at the pheromone gland or injection into the abdomen, deuterated III was detected in a pheromone extract by GC-MS analysis. Furthermore, the substrate selectivity of epoxidase and selective incorporation by the pheromone glands were examined by treatments with mixtures of the deuterated precursor and other hydrocarbons such as C19-C23 trienyl, C21 dienyl, and C21 monoenyl hydrocarbons. The 9,10-epoxy derivative of each alkene was produced, while the epoxidation of the C21 monoene was poorer than those of the trienes and diene. The low selectivity indicated that the species-specific pheromone of the H. cunea female was mainly due to the critical formation of the precursor of each component.

Studies on the poll glands of the one-humped camel in relation to reproductive activity. I. Seasonal morphological and histochemical changes.

The poll glands of the camel have been studied histologically and histochemically. An active gland, as observed between September and March, with a peak period in November and December, consists of sharply demarcated lobules separated by thin strands of connective tissue. Alveoli and proximal parts of excretory ducts are either lined with flat/simple cuboidal epithelium or with tall cells possessing distal protruding tips almost occluding the lumina; both segments, i.e. alveolus and proximal part of excretory duct, are therefore secretory. In the inactive gland there is an apparent increase in the interlobular stroma with massive deposition of collagenous fibres. The alveoli are lined with squamous to low columnar epithelium. The structural appearance in the castrate animal is essentially the same as that of the inactive gland except for its remarkable amount of interlobular tissue. Both delta 5-3 beta- and 17 beta-hydroxysteroid dehydrogenases have been demonstrated histochemically in the secretory portions of the gland. Their activity is restricted to the period between September and March and is comparatively highest during November and December. It is concluded that the morphological, enzymatic and secretory activities of the poll gland are correlated with testicular activity and rutting behaviour. It is suggested that the poll glands could be a source of sex pheromones.