MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.

WITH the development of effective anaerobic techniques, the composition of the human intestinal bacterial flora is now more clearly understood. Although the detailed composition is dependent on the nature of the diet (see, for example, Hill et al., 1971), in all cases studied the predominant faecal bacteria are those of the non-sporing strictly anaerobic groups. The metabolic significance of the gut flora is only slowly emerging, but a number of review articles (e.g., Smith, 1966;Scheline, 1968) attribute to it a wide range of reactions of toxicological interest.The involvement of intestinal bacteria in such reactions is usually inferred from indirect evidence-for example, the effect of antibiotic treatment on the metabolism of the drug, or the comparison of germ-free and conventional animals-and there are few direct studies using pure strains of bacteria. In particular, the metabolic activities of the non-sporing strictly anaerobic bacteria are virtually unknown.Many of the enzymes concerned in the metabolism of food additives and drugs are already well known to bacteriologists. For example, the /%glucosidase responsible for the hydrolysis of aesculin and salicin is also responsible for releasing the active agents from some cardiac glycosides (Lauterbach and Repke, 1960), from cascara sagrada (Fingl, 1965) and from cycasin (Spatz, McDaniel and Laqueur, 1966).In the process of detoxification, many compounds are conjugated by the liver to form, for example, glucuronides, and then excreted in the bile into the intestine (Smith, 1966). The glucuronides may then be hydrolysed either by enzymes of the gut mucosa or by enzymes of any bacteria present in the gut, releasing the aglycone, which may be reabsorbed and may be subsequently re-excreted by the liver into the gut.Escherichia coli is well known to produce a /3-glucuronidase (Buehler, Katzman and Doisy, 1951), but E. coli normally constitutes only a small proportion of the gut flora and there are no quantitative studies on the relative contributions to the glucuronidase activity of the gut contents by various bacteria. Similarly the contributions of the different bacterial species to the total activity of other glycosidases in the gut have not been assessed.We have therefore examined the gut flora of four laboratory animals commonly used in toxicological studies and estimated the activity of the glycosidases produced by the principal groups of intestinal bacteria. MATERIALS AND METHODSCharacterisation of the gut flora. All the animals examined were fed ad libitum-rats and mice on diet 41B (Oxoid), rabbits and guinea-pigs on diet SG1. The rats, mice and guineapigs were killed with chloroform and the rabbits by intravenous injection of air. Immediately (1971) 451Downloaded from www.microbiologyresearch.org by after death the abdomen was opened and the gastro-intestinal tract was ligatured at the oesophagus, at the duodenum at a point close to the stomach, at various points along the small intestine, at the ileo-caecal junction and at the rectum. After removal of t...

1. The metabolism of several alkyl- and alkoxy-substituted pyrazines in the rat has been investigated. 2. Alkyl substituted compounds were oxidized to the corresponding acids which were excreted in the urine as such or as their glycine cojugates. The extent of oxidation was reduced when two adjacent alkyl groups were present. In the latter case ring hydroxylation also occurred. Methoxy-substituted pyrazines underwent O-demethylation and ring hydroxylation. 3. Little or no biliary excretion of the pyrazines or their metabolites occurred. 4. Some preliminary results on the metabolism of 2-isobutyl-3-methoxy-pyrazine (the major characteristic flavour component of bell pepper) have been obtained. 5. For comparative purposes the metabolism of some similarly substituted pyridines was investigated.

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 M) to culture-activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50-M) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants-but not thiol redox active compounds-inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial-dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox-dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox-dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy. Supplementary material for this article can be found on the HEPATOLOGY website

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The metabolism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has recently been implicated in the mechanisms underlying ecstasy-induced neurotoxicity and hepatotoxicity. However, its potential role in ecstasy-induced kidney toxicity has yet to be investigated. Thus, primary cultures of rat and human renal proximal tubular cells (PTCs) were used to investigate the cytotoxicity induced by MDMA and its metabolites methylenedioxyamphetamine (MDA), alpha-methyldopamine (alpha-MeDA), and the glutathione (GSH) conjugates 5-(glutathion- S-yl)-alpha-MeDA and 2,5- bis(glutathion- S-yl)-alpha-MeDA. Cell viability was evaluated using the mitochondrial MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MDMA and MDA were not found to be toxic to either rat or human PTCs at any concentration tested (100-800 micro M). In contrast, 800 micro M alpha-MeDA caused 60% and 40% cell death in rat and human PTCs, respectively. Conjugation of alpha-MeDA with GSH resulted in the formation of even more potent nephrotoxicants. Thus, exposure of rat and human PTC monolayers to 400 micro M 5-(glutathion- S-yl)-alpha-MeDA caused approximately 80% and 70% cell death, respectively. 5-(Glutathion- S-yl)-alpha-MeDA (400 micro M) was more toxic than 2,5- bis(glutathion- S-yl)-alpha-MeDA to rat renal PTCs but equally potent in human renal PTCs. Pre-incubation of rat PTCs with either acivicin, an inhibitor of gamma-glutamyl transpeptidase (gamma-GT), or bestatin, an inhibitor of aminopeptidase M, resulted in increased toxicity of 5-(glutathion- S-yl)-alpha-MeDA but had no effect on 2,5- bis(glutathion- S-yl)-alpha-MeDA-mediated cytotoxicity. The present data provide evidence that metabolism is required for the expression of MDMA-induced renal toxicity in vitro. In addition, metabolism of 5-(glutathion- S-yl)-alpha-MeDA by gamma-GT and aminopeptidase M to the corresponding cystein- S-yl-glycine and/or cystein- S-yl conjugates is likely to be associated with detoxication of this compound. Thus, it appears that toxicity induced by thioether metabolites of ecstasy at the apical membrane of renal proximal tubular cells is the result of extracellular events, presumably redox cycling.

The ability of bacteria to catalyse the nitrosation of secondary amines has been investigated. It has been shown that this may be of importance in people with urinary tract infections living in areas where the concentration of nitrate in the drinking water is high.

The proliferating AR42J-B13 pancreatic cell line is known to respond to glucocorticoid treatment by producing foci of cells that express the liver-specific albumin gene. We demonstrate that this cell line also expresses liver-specific or liver-enriched functional cytochrome P450 proteins when stimulated to trans-differentiate into hepatocytes by glucocorticoid. These data suggest that this cell line has an unusual ability to trans-differentiate into functional hepatocytes and that it could be possible to generate a limitless supply of functional hepatocyte-like cells in vitro.