Characterisation of the chemical and biological properties of molecules with QSAR/QSPR and chemical grouping, and its application to a group of alkyl ethers.

This study presents a QSAR/QSPR modelling and chemical grouping (read-across) approach to provide information on the biological properties of a group of aliphatic ethers, with accurate biological predictions restricted to those physico-chemical and (eco)toxicological properties where the performance of QSAR/QSPR has been shown to be acceptable. The mathematical methods used ranged from multivariate regression models to PLS (partial least-squares), SVM (support vector machines) and Sammon's mapping. A novel grouping approach, based on a set of key descriptors, has been proposed to give a compact picture of the structural and biological properties of the compounds, and to provide a more mechanistic basis for the interpretations of chemical groups. Besides being a straightforward case study, the paper also exemplifies the capabilities and limitations of the methods in predictive toxicology on a more general level.

Chronic pulmonary effects of respirable methylene diphenyl diisocyanate (MDI) aerosol in rats: combination of findings from two bioassays.

Two independent bioassays are available which have examined the potential carcinogenicity of monomeric and polymeric methylene diphenyl diisocyanate (MDI) following long-term inhalation exposure in rats. These studies are not directly comparable, however, due to differences in design and conduct of the in-life phase, and differences in nomenclature used for some of the histopathological findings. This paper presents a definitive overview ofthe pulmonary toxicity of MDI developed following a thorough review of both investigations. As part of this process, the test materials and the designs of the studies were compared, and an in-depth review of lung lesions was conducted by an independent reviewing pathologist. This included the re-examination of the original lung slides, supported by an analysis of the exposure regimens, the results of which were used to develop an accurate profile of the doses received by the animals in the two studies. Histopathological findings were then combined with this information to give an overall dose-response curve for both studies as a whole. The range of total inhalation exposures to MDI was calculated as 559, 1972, 2881, 6001, 17,575 and 17,728 mgh/m3. Major pulmonary effects included increased lung weights together with bronchiolo-alveolar adenomas and hyperplasia, and interstitial fibrosis which occurred consistently in both studies, indicating a very similar qualitative response of the lungs to polymeric and monomeric MDI. The quantitative response of the lung was clearly dose-related in each study, and when the studies were considered as a whole a reasonable overall dose-response relationship was apparent for major lung lesions. Lung tumours (in low incidences) only occurred at the highest dose level in both studies (17,575 and 17,728 mgh/m3). For inflammatory and other non-neoplastic pulmonary changes, the lowest dose examined (559 mgh/m3) was regarded as a no-observed-adverse-effect-level for both polymeric and monomeric MDI. It was concluded that the results of the two studies could be combined to serve as a basis for human risk assessment of MDI.

Ninety-day feeding study in Fischer-344 rats of highly refined petroleum-derived food-grade white oils and waxes.

Subchronic 90-day feeding studies were conducted in male and female Fischer-344 (F-344) rats on highly refined white mineral oils and waxes representative of those used for food applications. The goal was to help clarify the mixed results found in other toxicity studies with laboratory animals. Seven white oils and 5 waxes were fed at dietary doses of 20,000, 2,000, 200, and 20 ppm and compared with control groups on untreated diet; toxicity was assessed at 90 days and also after a reversal period of 28 days and/or 85 days. Higher molecular-sized hydrocarbons (microcrystalline waxes and the higher viscosity oils) were without biological effects. Paraffin waxes and low- to midviscosity oils produced biological effects that were inversely related to molecular weight, viscosity, and melting point; oil type and processing did not appear to be determinants. Biological effects were more pronounced in females than in males. Effects occurred mainly in the liver and mesenteric lymph nodes and included increased organ weights, microscopic inflammatory changes, and evidence for the presence of saturated mineral hydrocarbons in affected tissues. Inflammation of the cardiac mitral valve was also observed at high doses in rats treated with paraffin waxes. Further studies are required to elucidate the mechanism for the responses observed and the relevance of these inflammatory responses in the F-344 rat to other species, including humans.

Development changes to gut microflora metabolism in mice.

Developmental changes in the activities of bacterial nitrate reductase, nitroreductase and beta-glucuronidase and their response to fermentable dietary fibre, were investigated in caecal contents from suckling mice (2-week-old) and in mice aged 4-24 weeks fed either a purified fibre-free diet or that diet supplemented with 5% (w/w) pectin. There was no apparent age-related trend common to the three enzymes studied. Nitrate reductase activity in the mice fed the fibre-free diet did not markedly alter with age. Pectin administration, however, was associated with a significant increase in nitrate reductase activity, particularly in 4-week-old mice. Nitroreductase activity exhibited an overall upward trend in mice from 2 to 12 weeks and thereafter decreased. Caecal beta-glucuronidase activity in mice increased sharply between 2 weeks and 4 weeks of age, thereafter not changing significantly until the 24th week. Pectin feeding had no consistent effect on activities either of nitroreductase or beta-glucuronidase. The changes in enzyme activities with age were not related to the concentration of bacteria in the caecum, which was highest in the 2-week-old mice. We conclude that the weaning is a period in which marked changes in caecal bacterial enzyme activities can occur.

Effects of Escherichia coli lipopolysaccharide on nitrate synthesis and on nitrosation of proline in rats.

Male Ola:SD rats were fed purified diets containing 5 or 20% lactalbumin as the protein source, with or without concomitant administration of Escherichia coli lipopolysaccharide (50-250 micrograms/kg, ip), and changes in 24-hr urinary nitrate excretion, plasma urea, plasma-nitrate pool size and 24-hr urinary nitrosoproline excretion were measured. Urinary nitrate and urinary 14C-nitrosoproline excretion (after oral [14C]proline administration) were significantly greater for rats receiving the high-protein diet compared with those on the low-protein diet. The co-administration of lipopolysaccharide increased nitrate excretion in both diet groups (although the increase was greatest (relatively) in the animals fed 5% lactalbumin), but did not significantly alter urinary nitrosoproline excretion by either group. Plasma urea concentrations and plasma-nitrate pool size were increased by a high-protein diet and/or lipopolysaccharide administration. These findings suggest that treatments which alter the availability of nitrate in vivo are not necessarily associated with increased nitrosation of proline.

Continuous culture of human faecal bacteria as an in vitro model for the colonic microflora.

To investigate the role of human gut bacteria in the metabolism of potentially reactive compounds we have developed an in vitro model of the human faecal microflora using a two-stage continuous culture inoculated with human faeces. The cultured bacterial population retained many of the bacteriological and biochemical characteristics of the flora present in the faecal sample used for inoculation. Obligate anaerobes were the predominant bacterial types found in vitro and included Bacteroides ovatus and Bifidobacterium adolescentis. A comparison of in vivo (faeces) and in vitro bacterial enzyme activities that are known to be involved in the biotransformation of potentially toxic compounds found the activities of hydrolytic enzymes to be similar but reductive enzymes exhibited higher activities in the continuous culture model. When substrates of the enzymes were added to the culture vessel, the enzymes were induced to varying extents. The short-chain fatty acid profile in the culture was almost identical to that in faeces with the order of abundance being the same in two systems. These results indicate that the continuous culture of faecal bacteria can provide a suitable model for studying bacterial interactions and biotransformation of the human colonic flora.

Modified mutagen activation in hepatic fractions from rats fed dietary rutin--interaction between gut flora and host metabolism.

Male Sprague-Dawley rats were fed a purified diet or one supplemented with the glycosidic plant flavonoid (+)rutin for 14 days. Rutin treatment significantly increased caecal bacterial beta-glucosidase activity (responsible for the conversion of rutin to the flavonoid quercetin) and there was an associated increase in the capacity of hepatic fractions (S-9) to activate the food pyrolysis products IQ, MeIQ and MeIQx to bacterial mutagens in vitro. Hepatic conversion of aflatoxin B1 to a mutagen was unaltered while in vitro activation of quercetin was significantly lower in tissue fractions from the rutin-fed rats compared with those from controls. Rutin treatment was without effect, however, on a number of hepatic cytochrome P-450-dependent mixed-function oxidase activities. The results suggest that products of bacterial metabolism of rutin formed in the hindgut may influence the activity of hepatic enzymes involved in the activation of certain classes of mutagen.

The influence of incubation pH on the activity of rat and human gut flora enzymes.

The activities of three bacterial biotransformation enzymes (beta-glucuronidase, beta-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6-8. All three enzymes were influenced by pH, as exemplified by beta-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6.6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with beta-glucuronidase, which showed maximal activity at pH 6.0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8.0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11,000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.

The role of gut micro-organisms in the metabolism of deoxynivalenol administered to rats.

1. Oral administration of deoxynivalenol (DON) to control rats resulted in the appearance of a de-epoxy metabolite in urine and faeces. 2. When DON was administered to rats treated with antibiotics to deplete their gut microflora there was very little excretion of radioactivity as the de-epoxy metabolite in faeces or urine. 3. Incubation of DON with a strictly anaerobic preparation of gut contents resulted in the progressive appearance of de-epoxy DON during a 24 h incubation period. 4. Incubation of DON with liver homogenate did not result in the appearance of the de-epoxy DON metabolite. 5. These results indicate that the presence of de-epoxy DON in rat excreta, following the oral administration of DON, is the result of metabolism by micro-organisms in the gut.

Developmental changes in hepatic activation of dietary mutagens by mice.

Metabolic activation of the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and aflatoxin B1 by female BALB/c mice of different ages (2-24 weeks) was investigated in vivo and in vitro using Salmonella typhimurium TA98 as the indicator organism. The in vivo activation of the three mutagens was investigated in 4- and 24-week-old mice using an intrasanguineous host-mediated assay. All three compounds showed reduced levels of activation with the older hosts. Hepatic S9 fractions from female mice of varying ages between 2 and 24 weeks were used in the in vitro mutagenicity assay. To achieve optimal activation to bacterial mutagens, 5% S9 was required for aflatoxin B1 and Trp-P-2 and 10% S9 for MeIQ; age of donor generally had little effect on the profile of these protein activation curves. Under these optimal conditions MeIQ and Trp-P-2 both exhibited, as before, age-dependent decreases in activation over a wide range of mutagen concentrations, however the in vitro activation of aflatoxin showed no consistent change with age. Spectrophotometric measurements of S9 cytochrome P-450 content showed a decrease in concentration with increasing age, but this was not sufficient to account for changes observed in hepatic mutagen activation. However, changes in the activities of certain cytochrome P-450 isoenzymes and cytosolic GSH-transferases, which in turn result in changes in the activation and detoxification capacity of the liver, would appear to explain age-dependent changes in the activity of mutagens in vivo.

Influence of starches of low digestibility on the rat caecal microflora.

1. Male Sprague-Dawley rats were fed on either a purified, fibre-free diet or a diet in which half the maize starch was replaced with uncooked amylomaize or potato starch (equivalent to 100 or 200 g amylase-resistant starch (ARS)/kg diet respectively). Changes in short-chain fatty acids (SCFA), pH, ammonia and a number of bacterial variables in caecal contents were then assessed. 2. Both ARS supplements decreased caecal content pH by approximately 1-2 units, with an associated reduction in ammonia concentration. Potato starch significantly decreased the concentration of SCFA in the hindgut, while amylomaize supplementation increased propionic and butyric acids but decreased the occurrence of minor, branched-chain fatty acids. 3. Caecal bacterial biotransformation activities (beta-glucosidase (EC 3.2.1.21), beta-glucuronidase (EC 3.2.1.31), reduction of p-nitrobenzoic acid, apparent ammonia formation) were consistently decreased by both ARS sources. 4. The results demonstrate that amylase-resistant carbohydrate altered toxicologically important functions in the large-intestinal flora of the rat.

Protein-related differences in the excretion of nitrosoproline and nitrate by the rat--possible modification of de novo nitrate synthesis.

Male Ola:SD rats were fed purified diets containing 5 or 20% lactalbumin as the source of protein, and the daily urinary excretion of nitrate and nitrosoproline was measured. Animals fed the high-protein diet consistently excreted more nitrate and nitrosoproline than littermates fed the low-protein ration, despite a similar, negligible amount of nitrate in both diets. Furthermore, whereas nitrite administration enhanced nitrosoproline excretion in both diet groups, nitrate administration increased nitrosamine output in the low-protein animals but did not affect nitrosation by rats given the 20% lactalbumin ration. Animals fed the 5% lactalbumin diet produced a smaller volume of urine than did the 20% diet group but other measurements of renal function were comparable for both treatments. The results suggest differences in endogenous nitrosation between rats fed diets high or marginal in protein, possibly reflecting decreased nitrate synthesis in the low-protein group.

An investigation of the endogenous formation of apparent total N-nitroso compounds in conventional microflora and germ-free rats.

The endogenous formation of apparent total N-nitroso compounds (ATNC) has been investigated in germ-free (GF) and conventional (CV) microflora rats as a function of the drinking-water nitrate concentration. ATNC levels were below the 40 micrograms (N-NO)/kg detection limit in the blood, liver, kidney, spleen and small intestine of all CV and GF rats. For the CV rats ATNC were detected in concentrations of up to 370 micrograms (N-NO)/kg in the large intestine and up to 50 micrograms (N-NO)/kg in the stomach and there was a significant positive correlation between ATNC formation and the drinking-water nitrate level. Comparison of these results with those from GF rats showed that the ATNC in the stomach and large intestine of the CV animals were formed by microbial action, most probably involving bacterial nitrate-reductase activity.

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora.

The activities of four bacterial biotransformation enzymes (beta-glucosidase, beta-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.

Dietary modification of intestinal bacterial enzyme activities--potential formation of toxic agents in the gut.

The bacterial population colonising the large intestine is able to metabolise a variety of ingested or endogenously produced substances to products, some of which possess toxic, mutagenic or carcinogenic properties. Dietary components, resistant to digestion and absorption in the upper alimentary tract, may influence these reactions by altering the environment of the gut or through the provision of nutrients to the flora. Evidence for the involvement of bacterial enzymes in the formation of toxic products in vivo has come largely from animal studies, particularly where fermentable plant cell-wall components are present in the diet. The role of diet in the modification of toxicologically important bacterial biotransformation processes will be discussed. Preliminary data will also be presented from a study demonstrating changes in the enzymic activity of the human faecal flora induced by pectin and bran. The significance of these changes to the disposition of chemicals in the gut will be discussed.

The effect of various dietary fibres on tissue concentration and chemical form of mercury after methylmercury exposure in mice.

The whole-body retention of mercury after exposure of BALB/c mice to methylmercury was measured in animals fed fibre-free, 5% pectin, 5% cellulose or 5, 15 or 30% wheat bran diets. The rate of elimination of mercury was dependent on the diet fed, with dietary bran increasing the rate of elimination. The incorporation of 15 or 30% bran in the diet of the mice decreased the total mercury concentration in the brain, blood and small intestine, although the effects were significant only in those animals on 30% bran diet. The fibres had little effect on mercury levels in other tissues. The proportion of mercury found in the mercuric form was significantly greater in liver, kidneys and gut of mice fed bran. The results suggest that dietary bran may reduce the levels of mercury in the brain after methylmercury exposure and may therefore reduce the neurotoxic effects of the organomercurial. We suggest that wheat bran exerts its effects on mercury retention and brain level via a modification of the metabolic activity of the gut microflora.

The use of continuous flow systems for studying the metabolic activity of the hindgut microflora in vitro.

To investigate the involvement of bacterial enzyme activities in the biotransformation of xenobiotic compounds, we have developed a simulation of the rat hindgut microflora in vitro. This mixed bacterial population exhibits many similarities to the native rat flora, and the diversity of bacterial species and the activity of a number of hydrolytic and reductive enzymes (e.g. azoreductase, beta-glucosidase, beta-glucuronidase, nitrate reductase and nitroreductase) are reproduced in the culture at levels similar to those found in the large intestine. The flora have been found to respond to an anutrient (cyclamate) or to host products (bile acids) with changes in enzyme activity, and to metabolize the azo dye Brown HT to metabolites qualitatively similar to those found in the faeces after oral administration to the rat. The experiments demonstrate that the bacterial population of the large intestine of the rat may be successfully cultured in vitro and provides and alternative to animal studies for the investigation of foreign compound metabolism by the flora.