Increased cytogenetic damage detected by FISH analysis on micronuclei in peripheral lymphocytes from alcoholics.

Alcohol abuse greatly increases the risk of various malignancies, including cancer of the liver and digestive tract. Although it is thought that this may be due, at least partially, to the mutagenic properties of ethanol, little is known about the genotoxic effects of ethanol in humans. We investigated the chromosomal damage in lymphocytes from 20 alcoholics and 20 controls using the micronucleus (MN) assay combined with fluorescence in situ hybridization (FISH) with a pancentromeric DNA probe capable of differentiating centromere positive (C+) from centromere negative (C-) MN. The frequency of MN in binucleate lymphocytes was significantly higher in alcoholics than in controls (12.0 +/- 5.4 and 7.6 +/- 1.6, respectively; P: < 0.05). FISH revealed significantly higher frequencies of C+ MN in alcoholics than in controls (8.2 +/- 4.8 and 3.4 +/- 1.4, respectively; P: < 0.05). In the alcoholics, no association was found between years of alcohol abuse and frequency of MN or C+ MN. However, age influenced MN and C+ MN frequency both in alcoholics and controls. These results indicate that alcohol abuse may well induce chromosome loss in humans, suggesting a possible aneugenic mechanism of alcohol. This effect could contribute to the health hazards related to alcoholism such as cancer risk.

Extractive analysis of aluminum traces in dialysis solutions.

A very sensitive procedure for the fluorimetric determination of aluminum traces in dialysis solutions by means of Mordant Red 19 dyestuff is described with the extraction of the Al complex in isobutylmethylketone. The experimental conditions were studied, in order to obtain the best extraction yield. The emission intensity of the metal chelate, extracted in the organic layer, was measured at 549 nm, exciting at 485 nm. Linearity between emission intensity and Al concentration was found in the 1-30 ng/ml range. The limit of detection was 0.25 ng/ml. The method resulted to be suitable for the determination of Al traces in commercial dialysis solutions for toxicological purposes.

Fluorimetric determination of aluminum traces in hemodialysis solutions using Mordant Red 19.

A sensitive and accurate method for the spectrofluorimetric determination of trace levels of aluminum in hemodialysis solutions using Mordant Red 19 as the complexation reagent has been developed. The optimal experimental conditions for the concentration of fluorimetric reagent, pH, temperature, and the specific type of matrix are reported. The emission of the fluorescent metal chelate was measured at 555 nm, excitation at 478 nm. Linearity between emission intensity and aluminum concentration was found in the 2-20 ppb range in standard aluminum solutions. Limit of detection was 0.4 ppb. The aluminum amounts in some commercial hemodialysis solutions were determined by means of the extrapolation method. The proposed method proved to be suitable in terms of sensitivity and accuracy for the determination of aluminum in dialysis fluids.

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate.

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague-Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 mumol-1 min-1 100 g-1 body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrinkage were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features.

Metabolism, pharmacokinetics, and activity of a new 6-fluoro analogue of ursodeoxycholic acid in rats and hamsters.

BACKGROUND/AIMS: The effectiveness of ursodeoxycholic acid in treating biliary liver diseases is limited by low bioavailability and moderate activity. A new analogue of ursodeoxycholic acid was synthesized with a fluorine atom in position 6 because this should have resulted in an analogue more hydrophilic than ursodeoxycholic acid but with similar detergency. METHODS: After synthesis, detergency, solubility, and lipophilicity of the 6-fluoro analogue in aqueous solution were determined and compared with those of natural analogues. Stability toward 7-dehydroxylation was assessed in human stools, pharmacokinetics and metabolism were evaluated in bile fistula rats and hamsters, accumulation in bile with long-term feeding was assessed in the hamsters, and the ability to prevent the hepatotoxic effects of taurochenodeoxycholic acid was evaluated in bile fistula rats after intraduodenal coinfusion. RESULTS: 6-Fluoro-ursodeoxycholic acid was more stable than its parent molecule toward 7-dehydroxylation, it was efficiently secreted in bile, and its total recovery was very high. With long-term administration of 6-fluoro-ursodeoxycholic acid, taurine and glycine amidates accounted for more than 60% of the total biliary bile acids (15% ursodeoxycholic acid). The 6-fluoro analogue prevented the hepatotoxic effects of taurochenodeoxycholic acid. CONCLUSIONS: The results suggest that 6-fluoro-ursodeoxycholic acid has considerable potential as a pharmaceutical agent in the treatment of cholestatic liver disease.

New 6-substituted bile acids: physico-chemical and biological properties of 6 alpha-methyl ursodeoxycholic acid and 6 alpha-methyl-7-epicholic acid.

New analogs of ursodeoxycholic acid and 7-epicholic acid containing a 6 alpha-methyl group were synthesized, and their physico-chemical properties were studied and compared with those of their natural analogs. The 6 alpha-methyl group slightly increases the lipophilicity and slightly lowers the critical micellar concentration with respect to the corresponding natural analogs. Simulated bile 50% enriched with 6 alpha-methyl ursodeoxycholic acid, with a total bile acid/phospholipid ratio of 10/1, demonstrated a higher cholesterol-holding capacity and a faster cholesterol gallstone dissolution rate with respect to ursodeoxycholic acid, while 6 alpha-methyl-7-epicholic acid and 7-epicholic acid were much less efficient in these processes. The 6 alpha-methyl analogs were highly stable toward 7-dehydroxylation when incubated with human stool in anaerobic conditions. Their transport, metabolism, and effect on biliary lipid secretion were evaluated both in rats and hamsters after acute intravenous and intraduodenal infusion at a dose of 10 mumol/min per kg. In both species, 6 alpha-methyl ursodeoxycholic acid is efficiently secreted in bile, with a cumulative recovery similar to that of ursodeoxycholic acid. The only metabolites of 6 alpha-methyl ursodeoxycholic acid identified were its glycine and taurine amidated forms. 6 alpha-Methyl-7-epicholic acid was efficiently secreted into bile when infused intravenously, and to a lesser extent when infused intraduodenally, in both rats and hamsters; it was secreted in bile as amidate and also as free acid. When 6 alpha-methyl ursodeoxycholic acid, 6 alpha-methyl-7-epicholic acid, ursodeoxycholic acid, and 7-epicholic acid were chronically administered to hamsters (for 3 weeks, at a dose of 50 mg/kg per day) their accumulation in gallbladder bile was, respectively, 25.1%, 4.0%, 15.2%, and 3.4% of the total bile acids. In conclusion, of the two analogs, only 6 alpha-methyl ursodeoxycholic acid shows potential as a cholesterol gallstone-dissolving agent. In this regard, its most important properties are moderate lipophilicity, good metabolic stability, and better conservation in the enterohepatic circulation, with respect to ursodeoxycholic acid.

Mechanism of genotoxicity and electron density distribution by NMR of 5-nitro-3-thiophenecarboxamides, a novel group of direct-acting mutagens in Salmonella typhimurium.

The mutagenic activity of 23 5-nitro-3-thiophenecarboxanilides and of 5-nitro-3-thiophenecarboxamide, the prototype, (NTCAs) have been evaluated in the Ames test on Salmonella typhimurium strains TA100 ad TA98 with and without metabolic activation. Effects of different substituents (electron-donating and electron-withdrawing) were studied to evaluate structural features that affect the metabolism and the bacterial mutagenic potency. All the derivatives were direct-acting mutagens, the mutagenic potency ranging from 0.7 to 142 revertants (rev.)/nmol in TA100 and from 0.09 to 68 rev./nmol in TA98 strain. Results obtained with strains TA98NR and TA98/1,8-DNP6 indicated that the mutagenic activity was largely dependent on bacterial nitroreductase, whereas the O-acetylation step was not critical for mutagenic potency. Superoxide (O2-.) and hydroxyl (OH.) scavengers as well as other radical scavengers and enzymes inhibited NTCAs mutagenicity to different extents. In particular, O2-. seemed to be involved in NTCAs mutagenicity, showing a free radical pathway for NTCA metabolism. [1H]- and [13C]NMR data indicated that the effects of different substituents on genotoxicity are probably not exerted on the electron density distribution. The importance of factors such as extent of nitration, reduction potential, orientation of nitrosubstituent and planarity of the molecule are discussed.

Murine alpha/beta interferons inhibit benzo(a)pyrene activation and mutagenesis in mice.

In addition to their antiviral and immune regulatory properties, interferons (IFNs) are known to depress hepatic cytochrome P450-dependent metabolism. As many chemical mutagens and carcinogens require bioactivation by the mixed-function monooxygenase (MFO) system in order to be genotoxic, a combined genetic and biochemical approach was used to establish whether IFNs could inhibit the activation of benzo(a)pyrene (BaP) to the ultimate clastogenic metabolite(s) in vivo. Treatment of mice with murine IFN-alpha/beta depressed cytochrome P450 content, as well as ethoxyresorufin O-deethylase activity (EROD), as a probe of class IA1 P450 isozymes, for 24 hrs and delayed the attainment of normal levels to approximately 30 hrs. After IFNs plus BaP treatment, EROD activity showed a reduction up to 70% after 24 hrs with an enhancement in activity at 30 hrs. A positive correlation exists between the rate of inhibition of oxidative BaP hepatic metabolism and inhibition of clastogenic effects in vivo, as scored in the bone marrow chromosome aberration assay.

Pharmacokinetics of ursodeoxycholic acid in rat.

The pharmacokinetic behaviour and metabolism of ursodeoxycholic acid (UDCA) have been studied in the rat. After oral administration of both 3H-labelled (4 muCi/kg body wt) and unlabelled (20 mg) UDCA, UDCA appeared in serum almost entirely in conjugated form (taurine conjugated); UDCA was present in bile mostly as taurine conjugated; the more relevant metabolite is 3 alpha,6 alpha, 7 beta-trihydroxycholanoic acid which represents 10% of the total bile acid pool. UDCA increased bile flow and selectively decreased biliary cholesterol secretion, while phospholipid secretion was unaffected. Faecal UDCA excretion was 15-20% while the urinary extraction was 1.5% during 24 h. The data show that UDCA, when administered in high dose, is promptly secreted into bile almost entirely metabolized to tauroursodeoxycholic acid, where it (1) desaturates the cholesterol in bile, (2) exerts choleretic properties.

Study of the lipophilic character of a series of beta-carbolines.

The lipophilic character of a series of beta-carbolines has been studied. The RM values were measured by means of a reversed-phase thin-layer chromatographic (TLC) technique and compared with the RM values obtained by high-performance TLC (HPTLC), the log k' obtained by high-performance liquid chromatography (HPLC), and the log P values. The best equation shows a very good linear relationship between our RM values and the classical log P values obtained using an octanol-water system. The choice of a pH of 13.0 for the TLC system allowed the measurement of the RM values of molecules in their non-ionized form. The deviations from the linear relationship shown by the RM(HPTLC) and log k' values of two compounds were due to the fact that both compounds were at least partially ionized at the pH of 7.0 at which the HPTLC and HPLC determinations were carried out.

Toxic and DNA-damaging activities of the fungicides mancozeb and thiram (TMTD) on human lymphocytes in vitro.

The cytotoxic and mutagenic effects of the fungicides mancozeb and thiram were studied using human peripheral blood lymphocytes cultured in vitro with or without an S-9 mix microsomal metabolizing system. The results obtained suggested that the chemicals caused dose-dependent inhibition of thymidine uptake and unscheduled DNA synthesis on both resting and proliferating lymphocytes in the absence of the S-9 mix. In the presence of the S-9 mix, only thiram showed mutagenic activity by eliciting unscheduled DNA synthesis and a significantly higher frequency of sister chromatid exchanges than did controls.

NADPH-generating system: influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions.

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisole O-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and beta-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures. In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD). Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH. At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D7 strain of Saccharomyces cerevisiae.

Effect of repeated ether anesthesias on the mono-oxygenase system of rat liver S-9 fraction.

This study was designed to investigate the effect of ether anesthesia in rats, before i.p. injections to induce the mono-oxygenase enzyme system, on biochemical properties of liver S9 fractions. Aminopyrine N-demethylase and rho-nitroanisole O-demethylase activity levels, their stability, and lipid peroxidation were determined in S9 fractions after etherization (about 1 min in ether vapor chamber daily for 3 consecutive days, before i.p. injections of Na-phenobarbital and beta-naphthoflavone) and compared with controls receiving the same injections without etherization. The activities were slightly (but not significatively) enhanced after this treatment, but stability was markedly and significatively greater after 1 h of incubation in the conditions of the liver microsomal assay (+ 14.8% and + 74.7%, respectively); lipid peroxidation was strongly and significatively depressed (-76.0%). Etherization sufficient to kill the animals on the 4th day resulted in equally active but less stable S9 fraction enzymes. Dimethylnitrosamine (as a standard premutagen) was assayed with the D7 strain of Saccharomyces cerevisiae using S9 fractions obtained from both anesthetized and nonanesthetized rats. According to biochemical data, results obtained with S9 from partially anesthetized rats were comparable with the conventional ones (S9 from nonanesthetized rats). On the contrary, the use of more prolonged ether anesthesia, including one on the day the animals are killed, gives S9 fraction significantly less effective. We conclude that if brief etherizations are used, for i.p. injections only, the S9 fractions obtained are entirely satisfactory and the procedures involved in production are simplified; the additional animal treatment (etherization) must be specified.

Kinetics of uptake and biliary excretion of benzo(alpha)pyrene and mutagenic metabolites in isolated perfused rat liver.

An isolated liver perfusion system was used as a simplifying tool to study the metabolism and excretion of benzo(alpha)pyrene (BP) as a prototype carcinogen/mutagen. Phenobarbital (PB) was used to induce liver microsomal enzymes in Sprague-Dawley male rats prior to isolated liver perfusion. Control livers were run simultaneously using generally tritiated (G-3H)BP/BP as substrate in the perfusion medium. Both biliary excretion and liver weight were increased in the induced compared to control liver, but biliary flow when corrected for liver weight is statistically the same for both control and PB-induced livers. The excretion rat of radioactivity in the bile is always higher for PB-induced than for control liver (maximum radioactive excretion at 1 hr). There is a more rapid radioactivity removal in the liver perfusion medium for PB-induced than for control livers. Data are explained by increased metabolism of BP in induced liver leading to the presence of more polar metabolites undergoing preferential biliary excretion than in the control liver. Results support in vivo experimental data. Extracts from liver and bile were tested for microbial mutagenicity by the Ames test (TA 100) after TLC separation. The control liver shows virtually no mutagenicity in bile, only in TLC fractions from the liver. The PB-induced liver shows significant mutagenicity in several TLC fractions in both bile and liver. The net effect of induction is to produce more mutagenic metabolites of BP, excreted in the bile, and presenting a significant exposure of carcinogens/mutagens, and consequent hazard to man.

Bile as a source of mutagenic metabolites produced in vivo and detected by Salmonella typhimurium.

The in vivo metabolic activation of several mutagenic compounds was assayed in the bile of rats, with Salmonella typhimurium as the indicator organism. It was determined that for some compounds, particularly the aromatic amines, a substantial percentage of the compound was excreted into the bile as either a nonmutagenic glucuronide conjugate or a mutagenic metabolite of the compound. Relatively low doses of the chemical were detected, and it was possible to follow the excretion pattern of the compounds over the collection period.