Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture.

Birds are uricotelic, and because they excrete urate by renal tubular secretion, they provide a convenient model for examination of this process. Primary monolayer cultures of the isolated renal proximal tubule epithelium from the domestic chicken, Gallus gallus L., were mounted in Ussing chambers where several substrates/inhibitors of renal organic anion transporters were tested for the sidedness and specificity of their effects on transepithelial urate transport. Transepithelial electrical resistance, electrical potential and sodium-dependent glucose current were monitored to detect nonspecific effects. Under control short-circuited conditions the ratio of unidirectional fluxes of [(14)C]urate was found to be 3:1. Active net secretion was specifically inhibited by 1 mmol l(-1) probenecid and 10 mmol l(-1) para-aminohippuric acid (PAH). Bromocresol Green, cimetidine, nocodozole, cytochalasin D and ouabain also inhibited secretion but were toxic. Interstitial-side lithium (5 mmol l(-1)) and glutarate (1 mmol l(-1)) specifically blocked transport, but 10-100 micromol l(-1) glutarate had no effect. Interstitial estrone sulfate (ES) stimulated urate secretion at 10 micromol l(-1) but was inhibitory at 500 micromol l(-1). Active PAH secretion (5:1 flux ratio) was inhibited 34% by 330 micromol l(-1) urate. ES (500 micromol l(-1)) blocked the remainder. From the lumen side, glucose-free, Cl(-)-free and high K(+) (30 mmol l(-1)) solutions, or an alkaline pH of 7.7 had no effect on urate transport and neither did several compounds known to be uricosuric. Lumen-side methotrexate (500 micromol l(-1)) and MK571 (20 micromol l(-1)) strongly inhibited urate secretion. MK571 had no effect from the interstitial side. RT-PCR revealed mRNA for OAT1-, OAT3-, MRP2- and MRP4-like organic anion transporters in chicken proximal epithelium.

Polycyclic aromatic hydrocarbons with bay-like regions inhibited gap junctional intercellular communication and stimulated MAPK activity.

Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens. A considerable amount of research has been devoted to predicting the genotoxic, tumor-initiating potential of PAHs based on chemical structure. However, information on the correlation of structure with the non-genetoxic, epigenetic events of tumor promotion is sparse. PAHs containing a bay or bay-like region were shown to be potent inhibitors of gap-junctional intercellular communication (GJIC), an epigenetic event involved in the removal of an initiated cell from growth suppression. We tested the epigenetic toxicity of PAHs containing bay-like regions by comparing the effects of methylated vs. chlorinated isomers of anthracene on the temporal activation of mitogen-activated protein kinase (MAPK) and the regulation of GJIC. Specifically, we used anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9,10-dimethylanthracene, 1-chloroanthracene, 2-chloroanthracene, and 9-chloroanthracene. We determined the effect of these compounds on GJIC and on the activation of extracellular receptor kinase (ERK 1 and 2), a MAPK, in F344 rat liver epithelial cells. Results showed that bay or bay-like regions, formed by either chlorine or a methyl group, reversibly inhibited GJIC at the same doses, time, and time of recovery, whereas the linear-planar isomers had no effect on GJIC. Similarly, the GJIC-inhibitory isomers also induced the phosphorylation of ERK 1 and ERK 2, while the non-inhibitory isomers had no effect on the activation of these MAPKs. MAPK activation occurred 10-20 min after the inhibition of GJIC, which indicates that MAPK is not involved in the initial regulation of GJIC; instead altered GJIC may be affecting MAPK activation. The present study revealed that there are structural determinants of PAHs, which clearly affect epigenetic events known to be involved in the non-genetoxic steps of tumor promotion. These events are the release of a cell from growth suppression involving the reduction of GJIC, followed by the activation of intracellular mitogenic events.

Mutagenicity testing of organic extracts of diesel exhaust particles after spiking with polycyclic aromatic hydrocarbons (PAH).

In the present study, spiking was used as a strategy to evaluate the mutagenicity of individual compounds in a mixture. Mutagenicity of individual polycyclic aromatic hydrocarbons (PAH) was evaluated in an organic extract of diesel exhaust particles (DEP). The particles were extracted with dichloromethane (DCM). After replacing DCM with dimethylsulphoxide (DMSO), the extract was spiked with four individual PAH: benzo(a)pyrene, benzo(a)anthracene, pyrene and fluoroanthene. The PAH were added separately and in various combinations to the extract to determine the effects of each variable and to identify possible interactions between the individual PAH and between the PAH and the extract. The study was designed as a fractional factorial experiment with the five variables (the DEP extract and the four PAH), giving 16 (instead of 32) mixtures plus a triplicate centrepoint and background, i.e. a total of 20. The fractionated factorial design used in the present work supports a model with linear and interaction terms. The mixtures were tested for mutagenicity in the Ames assay using four strains of Salmonella typhimurium in the presence of rat liver xenobiotic enzymes (S9-mix). Projections to Latent Structures (PLS) was used to quantify the mutagenicity of each compound and possible interactions. The four individual PAH and the DEP extract acted additively in the Ames test with 10% S9-mix.

Tissue-selective uptake of pravastatin in rats: contribution of a specific carrier-mediated uptake system.

Previously we demonstrated that a hydrophilic HMG-CoA reductase inhibitor, pravastatin, was actively taken up by the liver via the 'multispecific anion transporter' using isolated rat hepatocytes (M. Yamazaki, H. Suzuki, M. Hanano, T. Tokui, T. Komai, and Y. Sugiyama, Am. J. Physiol., 264, G36-G44 (1993)). Such a carrier-mediated uptake of pravastatin may contribute to the liver selective inhibition of the cholesterol synthesis in vivo. To examine the early-phase tissue distribution of this drug, we carried out a pharmacokinetic and tissue distribution analysis of pravastatin in rats. After i.v. bolus administration of [14C]pravastatin, the time profiles of [14C]radioactivity in plasma and several tissues were determined to calculate the tissue uptake clearance (CLuptake). Among the tissues examined, liver accounted for the major uptake (CLuptake,liver = 22.8 mL min-1 kg-1), followed by kidney (CLuptake,kidney (GFR corrected) = 2.36 mL min-1 kg-1). Other tissues showed no significant uptake (less than 0.2 mL min-1 kg-1). After portal vein administration, the distribution to the liver became much larger than that to the kidney due to the extensive first-pass removal by the liver. The first-pass hepatic uptake ratio was estimated as 0.66. Administering a range of doses (0.4-400 mumol kg-1) intravenously, an increase in early-phase half-life and a decrease in CLuptake,liver were observed simultaneously at doses over 40 mumol kg-1. In addition, CLuptake,kidney decreased at doses over 4 mumol kg-1. The effect of DBSP or PAH co-infusion (i.e. typical substrates for the transport system for organic anions in liver and kidney, respectively) on the initial uptake of pravastatin was also examined. DBSP clearly inhibited both the hepatic and renal uptake; however, PAH did not reduce the hepatic uptake of pravastatin although it inhibited the renal uptake. The transport systems in liver and kidney are thus considered different, based on the different saturability and inhibitory effect of organic anions.

The carcinogenic potency of carbon particles with and without PAH after repeated intratracheal administration in the rat.

The role of carcinogenic PAH in soot- and carbon black-related lung tumour induction in rats was investigated after intratracheal administration of carbon blacks (CB) and two types of diesel soot (DS), either as original or as toluene extracted particles. The total particle dose per animal was 15 mg subdivided into 16-17 weekly applications. There was one vehicle control and two groups were treated with a total dose of either 30 or 15 mg pure BaP as positive control. The main tumour results were: (a) original DS induced a higher tumour rate than extracted DS; (b) the carcinogenic potency of extracted CB probably depends on the size of the primary carbon particles and on the specific surface area of the particles; (c) extracted DS covered with 11 micrograms BaP per mg carbon particles caused a lower lung tumour rate than original DS containing only 0.9 ng BaP per mg, but a variety of other PAH and NO2-PAH; (d) a total dose of 15 mg pure BaP caused a lung tumour rate very similar to that of 30 mg extracted DS, 15 mg original DS or 15 mg Printex 90T CB extracted or covered with approximately 29.5 micrograms BaP per mg CB.

Effects of operating variables on PAH emissions and mutagenicity of emissions from woodstoves.

As part of the Integrated Air Cancer Project, the U.S. Environmental Protection Agency (EPA) has conducted field emission measurement programs in Raleigh, North Carolina, and Boise, Idaho, to identify the potential mutagenic impact of residential wood burning and motor vehicles on ambient and indoor air. These studies included the collection of emission samples from chimneys serving wood burning appliances. Parallel projects were undertaken in instrumented woodstove test laboratories to quantify woodstove emissions during operations typical of in-house usage but under more controlled conditions. Three woodstoves were operated in test laboratories over a range of burnrates, burning eastern oak, southern yellow pine, or western white pine. Two conventional stoves were tested at an altitude of 90 m. One of the conventional stoves and a catalytic stove were tested at an altitude of 825 m. Decreasing burnrate increased total particulate emissions from the conventional stoves while the catalytic stove's total particulate emissions were unaffected. There was no correlation of total particulate emissions with altitude whereas total polynuclear aromatic hydrocarbon (PAH) emissions were higher at the lower altitude. Mutagenicity of the catalytic stove emissions was higher than emissions from the conventional stove. Emissions from burning pine were more mutagenic than emissions from oak.

Structure-nephrotoxicity relationships of glutathione pathway intermediates derived from organic solvents.

The nephrotoxicity of glutathione (GSH) pathway metabolites derived from toluene (TOL), styrene (STYR), bromobenzene (BB), acrylonitrile (ACLN) and 2-chloroacrylonitrile (CACLN) were compared with that of dichlorovinylcysteine (DCVC), using renal brush border and basal-lateral uptake parameters as indices. Cysteine conjugates and mercapturates of ACLN did not alter p-aminohippurate (PAH) uptake by renal tubule suspensions in contrast to its chlorinated homologue. O-, m- and p-conjugates of BB inhibited PAH uptake by 43-82%, the mercapturates showing more potency than corresponding cysteine conjugates. The TOL derivatives N-acetylbenzylcysteine curtailed PAH uptake but benzylcysteine was more effective. The GSH conjugate and mercapturate synthesized from STYR oxide were also active inhibitors but not its cysteine conjugate. Among all GSH pathway metabolites studied, only DCVC and phenylhydroxyethylglutathione, derived from STYR oxide, impeded the renal basal-lateral uptake of [14C]tetraethylammonium (TEA) while DCVC was the sole inhibitor of brush border transport events such as the uptakes of [3H]glutamate and [14C]alpha-methyl-D-glucoside. These data indicate that GSH conjugation represents a non-nephrotoxic detoxication pathway for ACLN. In contrast, GSH conjugation with 2-chloroacrylonitrile and with aromatic solvents like TOL, STYR, BB gives rise to nephrotoxic mercapturates which may be less potent but show more specificity for the organic anion transport system than DCVC.