Expression of cytochromes P450 and glutathione S-transferases in human prostate, and the potential for activation of heterocyclic amine carcinogens via acetyl-coA-, PAPS- and ATP-dependent pathways

Dietary factors appear to be involved in the high incidence of prostate cancer in "Westernized" countries, implicating dietary carcinogens such as heterocyclic amines (HAs) in the initiation of prostate carcinogenesis. We examined 24 human prostate samples with respect to their potential for activation and detoxification of HAs and the presence of DNA adducts formed in vivo. Cytochromes P450 1B1, 3A4 and 3A5 were expressed at low levels (<0.1-6.2 pmol/mg microsomal protein). N-Acetyltransferase (NAT) activities, using p-aminobenzoic acid (NAT1) and sulfamethazine (NAT2) as substrates, were <5-5,500 and <5-43 pmol/min/mg cytosolic protein, respectively. Glutathione S-transferases (GSTs) P1, M2 and M3 were expressed at 0.038-1.284, 0.005-0.126 and 0.010-0.270 microg/mg cytosolic protein, respectively; GSTM1 was expressed in all GSTM1-positive samples (0.012-0.291 microg/mg cytosolic protein); and GSTA1 was expressed at low levels (<0.01-0.11 microg/mg cytosolic protein). Binding of N-hydroxy-PhIP to DNA in vitro occurred primarily by an AcCoA-dependent process (<1-54 pmol/mg/DNA), PAPS- and ATP-dependent binding being <1-7 pmol/mg DNA. In vivo, putative PhIP- or 4-aminobiphenyl-DNA adducts were found in 4 samples (0.4-0.8 adducts/10(8) bases); putative hydrophobic adducts were found in 6 samples (8-64 adducts/10(8) bases). Thus, the prostate appears to have low potential for N-hydroxylation of HAs but greater potential for activation of N-hydroxy HAs to genotoxic N-acetoxy esters. The prostate has potential for GSTP1-dependent detoxification of ATP-activated N-hydroxy-PhIP but little potential for detoxification of N-acetoxy-PhIP by GSTA1. However, there were no significant correlations between expression/activities and DNA adducts formed in vitro or in vivo, DNA adducts in vivo possibly reflecting carcinogen exposure.     

Effects of an ethanol-gasoline mixture: results of a 4-week inhalation study in rats

The inhalation toxicity of an ethanol-gasoline mixture was investigated in rats. Groups of 15 male and 15 female rats were exposed by inhalation to 6130 ppm ethanol, 500 ppm gasoline or a mixture of 85% ethanol and 15% gasoline (by volume, 6130 ppm ethanol and 500 ppm gasoline), 6 h a day, 5 days per week for 4 weeks. Control rats of both genders received HEPA/charcoal-filtered room air. Ten males and ten females from each group were killed after 4 weeks of treatment and the remaining rats were exposed to filtered room air for an additional 4 weeks to determine the reversibility of toxic injuries. Female rats treated with the mixture showed growth suppression, which was reversed after 4 weeks of recovery. Increased kidney weight and elevated liver microsomal ethoxyresorufin-O-deethylase (EROD) activity, urinary ascorbic acid, hippuric acid and blood lymphocytes were observed and most of the effects were associated with gasoline exposure. Combined exposure to ethanol and gasoline appeared to exert an additive effect on growth suppression. Inflammation of the upper respiratory tract was observed only in the ethanol-gasoline mixture groups, and exposure to either ethanol and gasoline had no effect on the organ, suggesting that an irritating effect was produced when the two liquids were mixed. Morphology in the adrenal gland was characterized by vacuolation of the cortical area. Although histological changes were generally mild in male and female rats and were reversed after 4 weeks, the changes tended to be more severe in male rats. Brain biogenic amine levels were altered in ethanol- and gasoline-treated groups; their levels varied with respect to gender and brain region. Although no general interactions were observed in the brain neurotransmitters, gasoline appeared to suppress dopamine concentrations in the nucleus accumbens region co-exposed to ethanol. It was concluded that treatment with ethanol and gasoline, at the levels studied, produced mild, reversible biochemical hematological and histological effects, with some indications of interactions when they were co-administered.     

Formation and biochemistry of carcinogenic heterocyclic aromatic amines in cooked meats

Heteroyclic aromatic amines (HAAs) are a class of hazardous chemicals that are receiving heightened attention as a risk factor for human cancer. HAAs arise during the cooking of meats, fish, and poultry, and several HAAs also occur in tobacco smoke condensate and diesel exhaust. Many HAAs are carcinogenic and induce tumors at multiple sites in rodents. A number of epidemiologic studies have reported that frequent consumption of well-done cooked meats containing HAAs can result in elevated risks for colon, prostate, and mammary cancers. Moreover, DNA adducts of HAAs have been detected in human tissues, demonstrating that HAAs induce genetic damage even though the concentrations of these compounds in cooked meats are generally in the low parts-per-billion (ppb) range. With recent improvements in sensitivity of mass spectrometry instrumentation, HAAs, their metabolites, and DNA adducts can be detected at trace amounts in biological fluids and tissues of humans. The incorporation of HAA biomarkers in epidemologic studies will help to clarify the role of these dietary genotoxicants in the etiology of human cancer.     

Mechanisms of action of the carcinogenic heterocyclic amine PhIP

Formed during the cooking of meat, the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP) is mutagenic and carcinogenic. Although the metabolism and mutational effects of PhIP are well defined, the early cellular and genomic events by which it can induce neoplastic transformation are not yet fully characterised. These early cellular responses to genotoxic doses of PhIP were examined in a human mammary epithelial cell, MCF10A. Using Western blotting, PhIP was shown to induce expression of the DNA damage response proteins p53 and p21(WAF1/CIP1), and to inhibit cell growth while activating G1 cell cycle checkpoint, a consequence of PhIP-induced DNA damage. Using low doses of PhIP (previously shown to activate oestrogenic signalling), PhIP increased proliferation in the oestrogen receptor (ER)-negative MCF10A cell line and to activate the mitogen-activated protein kinase (MAPK) pathway. Inhibition of this pathway significantly reduced the PhIP-induced cell growth of MCF10A cells. The work presented here suggests that, further to its genotoxic properties, at levels close to human exposure PhIP stimulates cellular signalling pathways that are linked to the promotion and progression of neoplastic disease. It is possible that a combination of these DNA damaging and growth promoting properties provide a mechanism for the tumourigenicity of PhIP, and may be key determinants for the tissue specificity of PhIP-induced carcinogenesis.     

The identification of G-protein coupled receptors in sequence databases

In the race to identify high quality drug targets from the enormous amount of information generated by the Human Genome Project, many companies are focussing their efforts on proteins already known to be good therapeutic targets. The G-protein coupled receptors (GPCRs) represent the most common class of therapeutic target. In this review we explore the sequence attributes of GPCRs and outline how these might be applied to focused database searches for novel GPCRs and to efficiently evaluate these new gene products pharmacologically.     

Biogenic amine receptor gene expression in the ovarian tissue of the honey bee Apis mellifera

In the honey bee Apis mellifera loss of the queen from a colony induces increased levels of the biogenic amine dopamine in the brain of workers, and this elevation is correlated with ovary activation. In the present study we use real-time quantitative PCR to investigate expression of five biogenic amine receptor genes. We show that biogenic amine receptors are expressed in ovarian tissue, and that their expression is strongly influenced by the presence or absence of a queen in the colony. In contrast to the brain, where all three dopamine receptors are expressed, only two dopamine receptors are expressed in the ovaries, and their expression is strongly correlated with the reproductive status of workers. We conclude that biogenic amine receptors are expressed in the ovaries and are likely to be directly influential in the regulation of worker sterility in honey bees.     

Advances in the molecular characterization of tryptophan hydroxylase

The neurotransmitter serotonin has been implicated in numerous physiological functions and pathophysiological disorders. The hydroxylation of the aromatic amino acid tryptophan is rate-limiting in the synthesis of serotonin. Tryptophan hydroxylase (TPH), as the rate-limiting enzyme, determines the concentrations of serotonin in vivo. Relative serotonin concentrations are clearly important in neural transmission, but serotonin has also been reported to function as a local antioxidant. Identification of the mechanisms regulating TPH activity has been hindered by its low levels in tissues and the instability of the enzyme. Several TPH expression systems have been developed to circumvent these problems. In addition, eukaryotic expressions systems are currently being developed and represent a new avenue of research for identifying TPH regulatory mechanisms. Recombinant DNA technology has enabled the synthesis of TPH deletions, chimeras, and point mutations that have served as tools for identifying structural and functional domains within TPH. Notably, the experiments have proven long-held hypotheses that TPH is organized intoN-terminal regulatory and C-terminal catalytic domains, that serine-58 is a site for PKA-mediated phosphorylation, and that a C-terminal leucine zipper is involved in formation of the tetrameric holoenzyme. Several new findings have also emerged regarding regulation of TPH activity by posttranslational phosphorylation, kinetic inhibition, and covalent modification. Inhibition of TPH byl-DOPA may have implications for depression in Parkinson’s disease (PD) patients. In addition, TPH inactivation by nitric oxide may be involved in amphetamine-induced toxicity. These regulatory concepts, in conjunction with new systems for studying TPH activity, are the focus of this article.     

Histochemical Study of Mast Cells from the Thymus of Mice Receiving ACTH1-24

Synthetic ACTH1-24 analogue administered in a daily dose of 0.01 mg/kg decreased the number and size of mast cells and increased intracellular serotonin concentration. ACTH1-24 induced degranulation of young mast cells and release of undersulfated heparin. Correlation analysis showed that hormonal imbalance produced by ACTH1-24 was accompanied by redistribution of bioamines.