Effect of intestinal microflora on the urinary metabolic profile of rats: a (1)H-nuclear magnetic resonance spectroscopy study.

1. Analysis of urine by (1)H-nuclear magnetic resonance (NMR) spectroscopy is used to detect biochemical disturbances predictive of toxicological changes. Recent studies, using (1)H-NMR spectroscopy have suggested that Alderley Park rats can be classified as hippuric acid (HA) or m-(hydroxyphenyl)propionic acid (m-HPPA) excretors. Evidence exists for the role of intestinal microflora in the excretion of aromatic phenolic compounds including HA and m-HPPA. 2. We sought to investigate whether intestinal microflora contribute to the difference in excretion. Urinary HA and m-HPPA levels were monitored to characterize excretion over time. The effect of intestinal microflora on the (1)H-NMR spectrum was also investigated using antibiotics to sterilize the intestine. Finally, the levels of m-HPPA and phenylpropionic acid (a precursor for HA) were analysed in the caecum and colon (entire tissue, including contents). 3. Characterization confirmed the presence of HA and m-HPPA excretors; enquiries revealed that the rats were obtained from two floors within a barriered breeding unit. Housing the rats from the two floors together for 21 days resulted in comparable levels of HA and m-HPPA excretion demonstrating that the profiles are not stable. 4. Following antibiotic treatment, HA and m-HPPA excretion decreased, indicating that intestinal microflora contribute to the excretion of these compounds. Finally, m-HPPA levels were higher in the colon of rats that excreted m-HPPA whilst PPA was increased in the caecum and colon of rats that excreted HA. 5. These results demonstrate that the observed difference in HA/m-HPPA excretion is due to differences in the intestinal microflora.

The incorporation of radiolabelled sulphur from captan into protein and its impact on a DNA binding study.

Repeated administration of high doses of captan is known to produce tumours specifically in the duodenum of mice. Captan is not carcinogenic in the rat. In this study, DNA purified from the liver, stomach, duodenum and jejenum of mice dosed with 35S radiolabelled captan was found to contain radioactivity equivalent to Covalent Binding Indices in the range 38-91; that from the bone marrow had a CBI of 2.8. The distribution of radioactivity between the various tissues did not reflect the target organ specificity of captan. Attempts to further purify the DNA samples using caesium chloride gradients resulted in partial separation of the radioactivity from the DNA suggesting that covalent binding to the DNA may not have occurred. A study of the chemical breakdown of captan showed that captan is unstable, producing a variety of potentially reactive species containing sulphur. Evidence was further obtained to show that the sulphur of captan is incorporated into endogenous amino acids and protein. Hepatic DNA from mice dosed with 35S radiolabelled N-acetylcysteine, and two thiazolidine derivatives which are analogous to known metabolites of captan, was radiolabelled to a similar extent to that from captan treated mice. Furthermore, the DNA from each of these treatments had similar properties on caesium chloride gradients. It was concluded that the radioactivity associated with DNA in the captan DNA binding study was present in the low levels of protein which are always associated with purified DNA samples.

Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice.

Methylene chloride (MC) induced DNA damage in freshly isolated hepatocytes from mice and rats, which was detectable as single-strand (ss) breaks by alkaline elution. The lowest in vitro concentration of MC needed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower than for rat hepatocytes (30 mM), and is close to the calculated steady-state concentration of MC in the mouse liver (1.6 mM) at a carcinogenic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectable in hepatocyte DNA from mice which had inhaled 4000 p.p.m. MC for 6 h, but not in hepatocyte DNA from rats similarly exposed. In studies with hepatocytes cultured overnight in the presence of buthionine sulfoximine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjugation of MC with GSH is important in its activation of DNA-damaging species in the liver. The GSH pathway of MC metabolism produces two potential DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH2Cl). Formaldehyde is known to cause DNA ss breaks in cells. However, the lowest concentration of formaldehyde required to induce a significant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikely to be formed following in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That formaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activation system from mouse liver (S9 fraction). Formaldehyde was responsible for the DNA- protein cross-linking effect of MC, but did not cause the DNA damage leading to ss breaks. These DNA ss breaks are likely to be caused by GSCH2Cl. The results suggest a genotoxic mechanism for MC carcinogenicity in the mouse liver, and support the proposal that the observed species differences in liver carcinogenicity result from differences in the amount of MC metabolism via the GSH pathway in the target organ.