Stress- and growth-related gene expression are independent of chemical-induced prostaglandin E(2) synthesis in renal epithelial cells.

Cellular stress can initiate prostaglandin (PG) biosynthesis which, through changes in gene expression, can modulate cellular functions, including cell growth. PGA(2), a metabolite of PGE(2), induces the expression of stress response genes, including gadd153 and hsp70, in HeLa cells and human diploid fibroblasts. PGs, gadd153, and hsp70 expression are also influenced by the cellular redox status. Polyphenolic glutathione conjugates retain the ability to redox cycle, with the concomitant generation of reactive oxygen species. One such conjugate, 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), is a potent nephrotoxic and nephrocarcinogenic metabolite of the nephrocarcinogen, hydroquinone. We therefore investigated the effects of TGHQ on PGE(2) synthesis and gene expression in a renal proximal tubular epithelial cell line (LLC-PK(1)). TGHQ (200 microM, 2 h) increases PGE(2) synthesis (2-3-fold) in LLC-PK(1) cells with only minor (5%) reductions in cell viability. This response is toxicant-specific, since another proximal tubular toxicant, S-(1, 2-dichlorovinyl)-L-cysteine (DCVC), stimulates PGE(2) synthesis only after massive (68%) reductions in cell viability. Consistent with the ability of TGHQ to generate an oxidative stress, both deferoxamine mesylate and catalase protect LLC-PK(1) cells from TGHQ-mediated cytotoxicity. Only catalase, however, completely blocks TGHQ-mediated PGE(2) synthesis, implying a major role for hydrogen peroxide in this response. TGHQ induces the early (60 min) expression of gadd153 and hsp70. However, while inhibition of cyclooxygenase with aspirin prevents TGHQ-induced PGE(2) synthesis, it does not affect TGHQ-mediated induction of gadd153 or hsp70 expression. In contrast, a stable PGE(2) analogue, 11-deoxy-16, 16-dimethyl-PGE(2) (DDM-PGE(2)), which protects LLC-PK(1) cells against TGHQ-mediated cytotoxicity, modestly elevates the levels of gadd153 and hsp70 expression. In addition, catalase and, to a lesser extent, deferoxamine mesylate block TGHQ-induced gene expression. Therefore, although TGHQ-induced generation of reactive oxygen species is required for PGE(2) synthesis and stress gene expression, acute TGHQ-mediated increases in gadd153 and hsp70 mRNA levels are independent of PGE(2) synthesis.

Reactive oxygen species and DNA damage in 2-bromo-(glutathion-S-yl) hydroquinone-mediated cytotoxicity.

Exposure of renal proximal tubular epithelial cells (LLC-PK1) to the nephrotoxicants 2-bromo-6-(glutathion-S-yl)hydroquinone, 2-bromo-3-(glutathion-S-yl)-hydroquinone, and 2-bromo-(diglutathion-S-yl)hydroquinone caused DNA fragmentation and cytotoxicity. Viability measured by lysosomal neutral red accumulation was the most sensitive parameter of cytotoxicity, and preceded toxicity determined by either the mitochondrial MTT assay or by measuring intracellular lactate dehydrogenase activity. DNA fragmentation was detected as early as 15 min after exposure to 2-bromo-6-(glutathion-S-yl)hydroquinone (100 microM), 2-bromo-3-(glutathion-S-yl)hydroquinone (200 microM), and 2-bromo-(diglutathion-S-yl)hydroquinone (400 microM) and prior to other indices of toxicity. The ability of the cells to repair DNA damage was evident by the decrease in the extent of single strand breaks following removal of 2-bromo-3-(glutathion-S-yl)hydroquinone from the incubation medium. Moreover, inhibition of poly(ADP-ribose)polymerase with 3-amino-benzamide (10 mM), following exposure of LLC-PK1 cells to 0.5 mM 2-bromo-6-(glutathion-S-yl)hydroquinone or 2-bromo-(diglutathion-S-yl)hydroquinone, decreased cytotoxicity, indicating that DNA repair processes, activated in response to DNA damage, exacerbate toxicity. Treatment with the endonuclease inhibitor, aurintricarboxylic acid did not decrease cytotoxicity. A decrease in the cytotoxicity caused by 2-bromo-6-(glutathion-S-yl)hydroquinone and 2-bromo-(diglutathion-S-yl)hydroquinone was observed when cells were incubated with catalase or pretreated with deferoxamine (10 mM). The data suggest a mechanism whereby the conjugates generate hydrogen peroxide, and the subsequent iron-catalyzed generation of hydroxyl radicals causes DNA fragmentation and cytotoxicity.

Inhibition of gamma-glutamyl transpeptidase potentiates the nephrotoxicity of glutathione-conjugated chlorohydroquinones.

Administration of either 2,5-dichloro-3-(glutathion-S-yl)-1, 4-benzoquinone (DC-[GSyl]BQ) or 2,5,6-trichloro-3-(glutathion-S-yl)-1,4-benzoquinone (TC-[GSyl]BQ) to male Sprague-Dawley rats caused dose-dependent (50-200 mumol/kg; iv) renal proximal tubular necrosis, as evidenced by elevations in blood urea nitrogen (BUN), and in the urinary excretion of lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (gamma-GT) and glucose. Renal proximal tubular necrosis was also confirmed by histological examination of kidney slices prepared from DC-(GSyl)BQ- and TC-(GSyl)BQ-treated animals. Administration of the corresponding hydroquinone conjugates (DC-[GSyl]HQ and TC-[GSyl]HQ), prepared by reducing the quinones with a threefold molar excess of ascorbic acid, resulted in a substantial increase in nephrotoxicity. Moreover, in contrast to other glutathione (GSH)-conjugated hydroquinones, the nephrotoxicity of both DC-(GSyl)HQ and TC-(GSyl)HQ was potentiated when rats were pretreated with AT-125, an irreversible inhibitor of gamma-GT. Neither the quinone-GSH nor the hydroquinone-GSH conjugates caused any effect on liver histology or serum glutamate-pyruvate transaminase levels. The results suggest that coadministration of ascorbic acid with DC-(GSyl)BQ or TC-(GSyl)BQ decreases their interactions with extrarenal nucleophiles, including plasma proteins, and thus increases the concentration of the conjugates delivered to the kidney, and hence toxicity. Furthermore the ability of AT-125 to potentiate the nephrotoxicity of DC-(GSyl)HQ and TC-(GSyl)HQ suggests that metabolism of these conjugates by gamma-GT constitutes a detoxication reaction.

Cytotoxicity of S-(1,2,3,4,4-pentachlorobutadienyl)-L cysteine after apical and basolateral exposure of LLC-PK monolayers. Involvement of an amino acid transport system.

Glutathione conjugation and subsequent formation of cysteine conjugates are key steps in the nephrotoxicity of halogenated alkenes. In this metabolic activation several organs are involved. However little is known about the transporters responsible for the uptake of cysteine conjugates. Recent evidence suggest that amino acid transporters play a role in this uptake. Monolayers of LLC-PK1 cells, a kidney cell line, were exposed to S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-CYS). Cytotoxicity was used as a parameter for PCBD-CYS uptake. Basolateral exposure (1 h: 400 microM and 16 h: 25 microM) to PCBD-CYS resulted in a much higher aminooxyacetic acid inhibitable cytotoxicity than apical exposure, suggesting a preferential basolateral uptake of PCBD-CYS. Exposure to PCBD-CYS in the absence of sodium did not result in a decrease of the cytotoxicity, suggesting a sodium independency of the PCBD-CYS uptake. Amino acids and amino acid analogues were used as diagnostic compounds in the further identification of the PCBD-CYS transporter. In cis-inhibition experiments monolayers were co-incubated with PCBD-CYS and these diagnostic compounds during one hour. System L substrates such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) and cycloleucine did not inhibit cytotoxicity. D-Tryptophan, a model inhibitor of System T, caused a strong inhibition. System L has, in contrast to System T, a high sensitivity to trans-stimulation. Pre-loading the monolayers with the diagnostic compounds should cause an increase in cytotoxicity when System L is involved. Neither System L substrates such as BCH and cycloleucine nor D-tryptophan increased cytotoxicity. These results suggest a preferential basolateral uptake of PCBD-CYS in LLC-PK1 monolayers and involvement of an amino acid transporter with characteristics of System T.

Use of monolayers of primary rat kidney cortex cells for nephrotoxicity studies.

Kidney cells were isolated from rat kidney cortex and maintained in short-term monolayer cultures. A number of important parameters were studied in order to establish the usefulness of these cells for toxicity studies. Despite morphological differences between the cultured cells and similar cells in vivo, many relevant enzyme systems remained present and functional. Intracellular glutathione levels were stable up to 5 days in culture. The glutathione S-transferase activity during culture remained stable although at a lower level than in freshly isolated cells. Whereas rat kidney cytosol contained subunits 4, 7, 2 and 1, 3- and 5-day-old cultures contained glutathione transferase subunits 7, 2 and a small amount of subunit 1. Cytochrome P-450, although measurable in microsomes from freshly isolated cells, could not be determined after 1 day in culture. Organic anion transporters on the basolateral side and gamma-glutamyl transpeptidase on the apical side were present. Through cytotoxicity studies, beta-lyase activity could be demonstrated in the culture. Hence this monolayer culture system, which can be used in combination with filters, seems to be suitable for studying various mechanisms of nephrotoxicity.

Differential toxicity as a result of apical and basolateral treatment of LLC-PK1 monolayers with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine.

Monolayers of LLC-PK1 cells, a cell line with features typical of proximal tubular epithelial cells, were treated at the apical and basolateral side with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBD-GSH) and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-NAC). Apical treatment with PCBD-GSH (greater than 20 microM) resulted in cytotoxicity, which could be inhibited by acivicin and aminooxyacetic acid (AOAA), inhibitors of gamma-glutamyltranspeptidase (gamma GT) and beta-lyase respectively. In contrast apical treatment with PCBD-NAC was only toxic at high concentrations (greater than 850 microM), and this effect could hardly be inhibited by AOAA. Basolateral treatment of confluent LLC-PK1 monolayers, grown on porous membranes, with PCBD-GSH gave a much smaller response than apical treatment, consistent with the fact that gamma GT is predominantly present at the apical side. Basolateral treatment even with high concentrations of PCBD-NAC (1.1 mM) did not show an increase in cytotoxicity when compared to the effect after apical treatment. These results suggest the absence of an organic anion transporter, by which these conjugates in vivo are transported into the cells from the basolateral side. This supposition was substantiated in a study of transcellular transport of the model ions tetraethyl ammonium (TEA) and para-aminohippurate (PAH), in LLC-PK1 monolayers, grown as indicated above. No active PAH transport could be demonstrated, whereas an active TEA transport was present. The absence of an organic anion transporter limits the usefulness of LLC-PK1 cells for the study of nephrotoxicity of compounds, like PCBD-NAc, needing this transport to enter the cells. However, the finding of an active basolateral organic cation transporter, together with the presence of gamma GT, dipeptidase and beta-lyase, makes this system especially interesting for testing all compounds that use this transporter or these enzymes in order to elicit toxicity.