Localization of N-acetyltransferases NAT1 and NAT2 in human tissues.

Human acetyl coenzyme A-dependent N-acetyltransferase (EC 2.3.1.5) (NAT) catalyzes the biotransformation of a number of arylamine and hydrazine compounds. NAT isozymes are encoded at 2 loci; one encodes NAT1, formerly known as the monomorphic form of the enzyme, while the other encodes the polymorphic NAT2, which is responsible for individual differences in the ability to acetylate certain compounds. Human epidemiological studies have suggested an association between the "acetylator phenotype" and particular cancers such as those of the bladder and colon. In the present study, NAT1- and NAT2-specific riboprobes were used in hybridization histochemistry studies to localize NAT1 and NAT2 mRNA sequences in formalin-fixed, paraffin-embedded human tissue sections. Expression of both NAT1 and NAT2 mRNA was observed in liver, gastrointestinal tract tissues (esophagus, stomach, small intestine, and colon), ureter, bladder, and lung. In extrahepatic tissues, NAT1 and NAT2 mRNA expression was localized to intestinal epithelial cells, urothelial cells, and the epithelial cells of the respiratory bronchioles. The observed heterogeneity of NAT1 and NAT2 mRNA expression between human tissue types may be of significance in assessing their contribution to known organ-specific toxicities of various arylamine drugs and carcinogens.

Influences of surgical castration on the thymus of male rats.

In previous studies, we have shown that castration of Sprague-Dawley rats enhances thymic weight through puberty whilst sex steroids reduce the castration-induced hypertrophy. In the current study, we have confirmed that castration enhances thymic growth compared to age-matched intact controls. In addition, immunoassays were used to measure thymosin alpha1 and thymosin beta4 levels in sera from intact and castrate rats. Castrate animals displayed greater sera levels of thymosins compared to sera from intact animals. To test whether the enhanced thymic weight and increased levels of thymosins observed post-castration were able to influence immune function of castrate animals, concanavalin A was used in the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay to examine lymphocyte and thymocyte responses from both intact and castrate male rats. Responses of cells isolated from castrate rats demonstrated that lymphocytes and thymocytes were stimulated at low levels of concanavalin A (1.56-3.13 microg/ml for lymphocytes and 1.56 microg/ml for thymocytes) compared to the same cell types isolated from intact rats. Concentrations of concanavalin A ranging from 6.25 to 200 microg/ml produced no significant differences in response from intact and castrate animals.

Localisation of aryl sulfotransferase expression in human tissues using hybridisation histochemistry and immunohistochemistry.

To date, the laboratory has cloned seven unique human sulfotransferases; five aryl sulfotransferases (HAST1, HAST2, HAST3, HAST4 and HAST4v), an estrogen sulfotransferase and a dehydroepiandrosterone sulfotransferase. The cellular distribution of human aryl sulfotransferases in human hepatic and extrahepatic tissues has been determined using the techniques of hybridization histochemistry and immunohistochemistry. Human aryl sulfotransferase expression was detected in liver, epithelial cells of the gastrointestinal mucosal layer, epithelial cells lining bronchioles and in mammary duct epithelial cells.

Effects of castration on the lymphocytes of the thymus, spleen and lymph nodes.

It is well known that the thymus plays an important role in the development and maintenance of a competent immune system. The thymus atrophies with age, a process that is accelerated after puberty when there is elevation of serum sex steroid levels. We have used a panel of commercial monoclonal antibodies against various T and B cell surface markers to investigate the post-castration histological alterations in the thymus, spleen and lymph nodes of male Sprague-Dawley rats. Castration of 5-week-old male rats produced a significant increase in thymic weight (P < 0.05) compared to age-matched intact animals. The major observations from the immunohistochemical studies were post-castration elevations in staining for total T cells (MRC OX 19 and W 3/13), CD8 cells (MRC OX 8), B cells (MRC OX 12 and MARK-1) and cells bearing activation markers such as IL-2 receptor (MRC OX 39), transferrin receptor (MRC OX 26) and major histocompatibility class II antigen (MRC OX 6). These data suggest that following castration there is an increase in the ability of lymphocytes to respond to activation. As a result, there are elevated numbers of immature thymocytes within the thymus that undergo differentiation/maturation and consequently produce an increase in peripheral T and B cells.

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: functional and localization studies.

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N, O-acetylation and N, O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in some subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Effect of prepubertal gonadectomy and sex steroid treatment on the growth and lymphocyte populations of the rat thymus.

The age at which rats are most sensitive to prepubertal gonadectomy, in terms of thymic growth, was investigated. Gonadectomy enhanced thymic growth at each age; the greatest difference in thymic weight between gonadectomized and intact animals occurred in male rats gonadectomized at 5 weeks of age (64%) and in female rats gonadectomized at 4-5 weeks of age (43-46%). The effect of various synthetic sex steroids on growth and lymphocyte populations of the thymus in gonadectomized rats was examined. Diethylstilboestrol, 1 mg per animal, inhibited thymic growth by more than 35% in both males and females. Ethinyloestradiol, 40 micrograms per animal, inhibited thymic growth by 26% in males but by only 4% in females. Fluoxymesterone, 10 mg per animal, inhibited thymic growth by more than 46% in both sexes. Norgestrel, 12 micrograms per animal, had no effect on thymic growth. The synthetic steroids that significantly inhibited thymic growth decreased the intensity and altered the localization of staining for total T cells (antibody clone MRC OX 19), T helper cells and macrophages (W 3/5), T cytotoxic/suppressor cells (MRC OX 8) and B cells (MRC OX 12).