Transport and metabolism of glutathione conjugates of menadione and ethacrynic acid in confluent monolayers of rat renal proximal tubular cells.

Confluent monolayers of primary rat renal proximal tubular (RPT) cells were used to compare transepithelial transport and concomitant metabolism of two different glutathione (GSH) S-conjugates. For the GSH-conjugated quinone compound, [35S]GSH-conjugated menadione (MGNQ), no specific transepithelial transport was observed. Most likely, [35S]MGNQ passed the monolayer via paracellular leakage as the result of a reduction in monolayer integrity due to toxicity via extensive redox cycling of the quinone under the culture conditions. RPT cell monolayers metabolise MGNQ into a cysteinylglycine conjugate, which after intramolecular cyclization yields 2H-(3-glycinyl)-9-hydroxy-10-methyl-1,4-naphthothiazine. Acivicin, an inhibitor of gamma-glutamyltranspeptidase, inhibited the formation of this 1,4-napthothiazine adduct. The second product formed is 1,4-napthothiazine formed by loss of glycine via the action of dipeptidases. Similarly, no basolateral (B) to apical (A) transport of a GSH-conjugated alpha, beta unsaturated ketone, [14C]ethacrynic acid (EASG), occurred. However, net transport of [14C] radioactivity could be observed from A=>B direction. After 8 h, 23% of total [14C] radioactivity was transported from the apical to the basolateral chamber. In both the apical and basolateral chambers, free, unconjugated ethacrynic acid (EA) was observed. gamma GT-mediated metabolism of EASG to the much more unstable cysteinylglycine conjugate leads to relatively large amounts of free EA. Thus, the GSH conjugate is not transported but rather the cysteine adduct and/or free, unconjugated EA. In agreement with this, acivicin reduced A=>B transport of EASG and inhibited the formation of free EA. In conclusion, the confluent monolayers of RPT cells do not or no longer possess active basolateral transport systems for GSH conjugates. However, they are still quite useful for studying biotransformation reactions of thioether conjugates.

Cytotoxicity of 2-tert-butyl hydroquinone glutathione conjugates after apical and basolateral exposure of rat renal proximal tubular cell monolayers.

Confluent monolayers of renal proximal tubular (RPT) cells cultured on porous supports were used to investigate the cytotoxicity induced by glutathione conjugates of 2-tert-butyl-(1,4)-hydroquinone (SG-TBHQ) after apical and basolateral exposure. As judged from lactate dehydrogenase (LDH) leakage, cytotoxicity was observed after basolateral exposure of monolayers to 250 and 500 mum 2-tert-butyl-5-(glutathion-S-yl)hydroquinone (5SG-TBHQ). In these experiments, LDH leakage was 22.3 +/- 1.9 and 32.2 +/- 1.9%, respectively. Basolateral exposure of monolayers to 250 and 500 mum 6SG-TBHQ resulted in LDH leakage of 22.2 +/- 2.5 and 30.0 +/- 2.7%, respectively. The double conjugate, 2-tert-butyl-3,6-(diglutathione-S-yl)hydroquinone (3,6SG-TBHQ), was not toxic and LDH leakage was about control level (15.0%). Basolaterally located probenecid-sensitive organic anion transporters did not seem to play a part in the cytotoxic effect. However, when RPT cell monolayers were cultured in 24-well tissue culture plates, apical challenge with 250 mum 5SG-TBHQ induced a cytotoxic effect. In these experiments, LDH leakage was 33.5 +/- 0.6%. With these cells, inhibition of apical gamma-glutamyltranspeptidase (gammaGT) activity by acivicin, which was not toxic by itself, decreased 5SG-TBHQ-induced LDH leakage to 19.3 +/- 1.2%, whereas 6SG-TBHQ (also 250mum)-induced LDH leakage was increased to 55.3 +/- 1.0%. Co-incubation of RPT cells with SG-TBHQs in the presence of 1.5 mm ascorbic acid (AA) pointed to a pro-oxidant rather than an antioxidant effect of AA. Superoxide dismutase and catalase completely abolished SG-TBHQ-induced cytotoxicity. Since cultured RPT cells lack N-acetylation of cysteine conjugates, the N-deacetylation/N-acetylation ratio cannot have a vital role in renal toxicity of quinone thioethers in this in vitro system. It seems, therefore, that the cytotoxicity observed is mainly the result of extracellular redox cycling of SG-TBHQs. The lack of toxicity of 6SG-TBHQ after apical exposure could be due to detoxification by gammaGT-mediated cysteinylglycine- or cysteine-conjugate formation followed by cyclization, as shown for other related quinone glutathione conjugates. The relative importance of the observed effects for the in vivo situation is discussed.

In vitro metabolism of 5-fluoro-2-glutathionyl-nitrobenzene by kidney proximal tubular cells studied by 19F-NMR.

Proximal tubular biotransformation of the glutathionyl (GSH) conjugate derived from 2,5-difluoronitrobenzene (5-fluoro-2-glutathionyl-nitrobenzene) was studied by means of 19F-NMR. This method allows a direct and specific detection of the fluorinated metabolites formed, at a detection limit of 1 microM for an overnight NMR run. Incubation of a monolayer of LLCPK1 cells with 100 microM 5-fluoro-2-glutathionyl-nitrobenzene for 24 h showed that these cells metabolize this GSH conjugate into the corresponding cysteinylglycyl and cysteine conjugate. The expected N-acetylcysteine conjugate however was not formed as an endproduct. Additional experiments demonstrated the absence of N-acetyltransferase activity in LLCPK1 cell lysates incubated with FCysNB and also the rapid loss of this activity in isolated renal proximal tubular cells (RPT): freshly isolated RPT cells do convert FCysNB to FNAcNB as major metabolite but, upon cultivation, quickly lose this capacity. Since uptake of FCysNB might also be a limiting factor, we investigated transport of FCysNB from the apical to the basolateral side of the culture RPT cells. No indication for such transport was obtained. Thus, the absence of mercapturic acid formation in LLCPK1 cells and cultured RPT cells is the results of a decline in N-acetyltransferase activity and perhaps a deficient cellular uptake of the cysteine conjugate.

Differential detoxification of two thioether conjugates of menadione in confluent monolayers of rat renal proximal tubular cells.

This in vitro study describes proximal tubular toxicity of quinone thioethers after incubation of these compounds on either side of a renal proximal tubular cell (RPTC) monolayer. These cells were cultured to confluency on porous supports of tissue culture inserts, and the apically and basolaterally induced toxicity of two thioether conjugates of menadione (2-methyl-1,4-naphthoquinone) was investigated. As judged by lactate dehydrogenase (LDH) leakage, the glutathione (GSH) conjugate of menadione (MGNQ) was only toxic after basolateral challenge. However, after inhibition of gamma-glutamyl transpeptidase by acivicin, MGNQ was also toxic after apical challenge. The mercapturic acid of menadione [M(NAC)NQ] displayed cytotoxicity both after apical and basolateral challenge. From the basolateral side, MGNQ and M(NAC)NQ-induced cytotoxicity could be enhanced by inhibition of the organic anion transport system with probenecid. Inhibition of beta-lyase did not influence M(NAC)NQ-induced cytotoxicity. In addition, inhibition of intracellular N-deacetylation of M(NAC)NQ using paraoxon potentiated the observed toxic effect. Thus, it appears that the MGNQ and M(NAC)NQ-induced cytotoxicity is the result of extracellular events, presumably redox cycling. Putative uptake of the conjugates is likely to be associated with detoxification of these compounds.

Effect of glutathione and cysteine on apical and basolateral uptake and toxicity of CdCl(2) in kidney cells (LLC-PK(1)).

LLC-PK(1) kidney cells were cultured on filters, to investigate both apical and basolateral uptake and concomitant toxicity of cadmium. Two to four times as much (109)CdCl(2) was taken up through the basolateral side of the cell than through the apical side. Equally, toxicity was found to be consistently greater after basolateral exposure than after apical exposure. At a non-toxic concentration of 1 mum-CdCl(2), extracellular glutathione (GSH) (1 mm) and cysteine (1 mm) both lowered the rate and extent of uptake of Cd during the first 4 hr. This effect was more pronounced after basolateral than after apical exposure. A series of toxicity experiments was performed to investigate the time dependence of the protection by GSH and cysteine: after a 1-hr exposure to 200 mum both protected against toxicity; after 2 and 4 hr of exposure to 50 mum the protection of GSH was less pronounced than the effect of cysteine. As complex formation between Cd and GSH or cysteine is reversible, toxicity found in the presence of GSH and cysteine at higher concentrations or larger exposure times may very well be due to Cd ions released from the extracellular complex with these thiols. Thus, it seems that the toxicity is caused by the uptake of extracellularly available free ionic Cd.

Comparative toxicity and accumulation of cadmium chloride and cadmium-metallothionein in primary cells and cell lines of rat intestine, liver and kidney.

The protective role of metallothionein (Mt) in the toxicity of cadmium (Cd) is controversial, since Cd bound to Mt is more nephrotoxic than ionic Cd after parenteral exposure and less hepatotoxic than ionic Cd after oral exposure. This study compared the uptake and toxicity in vitro of CdCl(2) and two isoforms of rat cadmium-metallothionein (CdMt-1 and CdMt-2) using primary rat kidney cortex cells, primary rat hepatocytes, liver hepatoma cell line H-35, kidney epithelial cell line NRK52-E and intestinal epithelial cell line IEC-18. The molar ratio of Cd was 2.1 and 1.4 mol Cd/mol Mt for CdMt-1 and CdMt-2, respectively. Monolayer cultures were incubated for 22 hr with CdCl(2), CdMt-1 or CdMt-2 and Cd accumulation was examined at Cd levels of 0.25-10 muM-Cd. Cells exposed to CdCl(2) accumulated more Cd in 22 hr than cells exposed to an equimolar amount of CdMt. For CdCl(2) the Cd accumulation is directly related to the Cd concentration in the medium; however, for CdMt an increase in Cd concentration in the medium above 2 muM had no effect on the Cd accumulation in the cells. At Cd concentrations above 2 muM, therefore, the difference in Cd accumulation between CdCl(2) and CdMt was greater (5-6 times) than at concentrations below 2 muM (1-2 times). Cytotoxicity was examined in the Cd-concentration range from 0.25 to 100 muM by determining the lactate dehydrogenase (LDH) release in the medium and the neutral red uptake in the cells. Under these culture conditions CdCl(2) was at least 100 times more toxic than CdMt-1 or CdMt-2 in all cell types tested. Primary hepatocyte cultures were 10 times more sensitive (50% LDH release at 1-2 muM) to CdCl(2) intoxication than primary cultures of renal cortical cells or the intestinal cell line (50% LDH release at 10-20 muM). Hepatic and renal cell lines were less sensitive (50% LDH release at 20-35 muM) than the corresponding primary cultures. No difference in sensitivity towards CdMt-1 or CdMt-2 was found for the various cell types tested. To investigate the influence of the molar Cd ratio of CdMt on cytotoxicity, the Cd content of CdMt-1 (2.1 mol Cd/mol Mt) was artificially raised in vitro to 5 mol/mol Mt. Compared with native CdMt, CdMt with a high molar Cd ratio in primary renal cultures showed a 15% increase in LDH release at a Cd concentration of 1500 muM in the medium. In conclusion, exogenous CdMt is far less toxic than CdCl(2) to cell cultures in a serum-free medium. Whereas CdCl(2) in all cases showed dose-dependent Cd accumulation, Cd accumulation due to CdMt exposure in all cell types tested reached a plateau at medium Cd concentrations of 2 muM. The low cellular Cd uptake of CdMt and the corresponding low cytotoxicity supports previously reported results in vivo, showing that the difference in toxicity between CdMt and CdCl(2) is associated with a difference in Cd distribution.

Endod: safety evaluation of a plant molluscicide.

Toxicity tests, in accordance with the Minimal Data Requirements (Tier 1) of the OECD Guidelines for Pre-Market Chemicals, were conducted on a standard extract (Endod-S) from the unripe berries of Phytolacca dodecandra, a potent botanical molluscicide of potential importance in the control of schistosomiasis. In acute mammalian toxicity tests, except for the eye irritation toxicity test which indicated severe irritancy, all test results were classified as either nontoxic or slightly toxic. Eye protection is therefore recommended during berry crushing and handling of dry powders. Ecotoxicity tests indicated that Endod is no more toxic than currently recommended synthetic molluscicides; however, environmental fate and additional local ecotoxicity tests are recommended for nontarget aquatic organisms present in the endemic situations of field trials. Given these toxicological data and recognizing the need for an affordable, locally cultivated, botanical molluscicide, it is concluded that field trials of Endod in schistosomiasis control are now justifiable.

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.

Differential effects of raising and lowering intracellular glutathione levels on the cytotoxicity of allyl isothiocyanate, tert-butylhydroperoxide and chlorodinitrobenzene.

The influence of intracellular glutathione (GSH) on cytotoxicity was studied in vitro by altering the intracellular GSH level of Chang liver cells. Lowering the GSH level was achieved by treating cells with diethyl maleate (155 mum), while the intracellular GSH level was raised by cellular incubation with the monoethyl ester of glutathione (20 mm). The cytoxicity of three compounds that are known to react differently with GSH was tested using the cloning efficiency assay. Allyl isothiocyanate reacts reversibly with GSH and in previous work it was shown that the toxicity of the GSH conjugate was due to the release of the free isothiocyanate. In this study we found that changing the intracellular GSH level had no influence on the cytotoxicity of this compound. This points to the cell membrane as the primary target, although the possibility of GSH being involved as a transporting agent cannot be completely excluded. Chlorodinitrobenzene conjugates easily with GSH, and in bacterial assays it was found that depletion of intracellular GSH enhanced the mutagenic effect. After raising the intracellular GSH level in Chang cells this compound was found to be more toxic, and after lowering the GSH level less toxicity was found, indicating a role for GSH in the activation of chlorodinitrobenzene. For comparison tert-butylhydroperoxide was chosen, because it is known to be detoxified by GSH. Lowering the intracellular GSH level did indeed result in higher cytotoxicity, but after elevating the GSH level no increase in cloning efficiency was observed, indicating that there are more factors involved in tert-butylhydroperoxide cytotoxicity.

A co-cultivation system consisting of primary chick embryo hepatocytes and V79 Chinese hamster cells as a model for metabolic cooperation studies.

In this study the role of metabolic cooperation was investigated in a co-cultivation system consisting of primary chick embryo hepatocytes and V79 Chinese hamster cells. A morphological study showed that, in addition to the gap junctions formed between homologous cells, gap junctions were formed also between the primary chick embryo hepatocytes and the V79 Chinese hamster cells. The number of gap junctions present in this system decreased in the following order: hep.-hep., V79-V79, hep.-V79. Under control conditions this number was constant during a co-cultivation period of 48 h. The heterologous gap junctions allowed the passage of 3H-labelled hypoxanthine. Addition of 12-O-tetradecanoylphorbol-13-acetate inhibited this transfer in a dose-related way. Electron microscopical studies with sectioned material showed that inhibition of transfer was paralleled by the disappearance of all gap junctions. There was a remarkable difference between the response time of the different types of gap junctions. Those formed between V79 cells had disappeared after 20 min, whereas those formed between hepatocytes had disappeared after 12 h. The heterologous gap junctions behaved more or less like those between hepatocytes. After exposure times longer than 7 h the transfer of [3H] hypoxanthine was partly restored and morphologically the gap junctions reappeared. When the V79 cells were pretreated with mitomycin C no recovery of intercellular communication was observed, indicating that the adaptation phenomenon is related to the mitotic index of the cells. Dimethylbenzanthracene inhibited the transfer of labelled nucleotides and may be the first example of an indirectly acting inhibitor of intercellular communication.

Ultrastructural characterization of leucocytes in the pronephros of carp (Cyprinus carpio, L.).

In the pronephros of carp (Cyprinus carpio, L.) the following cells were found and ultrastructurally characterized: erythrocytes; lymphocytes and plasma cells; thrombocytes; neutrophilic, eosinophilic and basophilic granulocytes; phagocytic reticular cells and monocytes and non-phagocytic reticular cells. The cells could be separated on a Percoll continuous density gradient and relatively pure fractions could be obtained. In vitro phagocytosis of bacteria (Bacillus megaterium) was found in monocytes and neutrophilic granulocytes, while basophilic and eosinophilic granulocytes engulfed bacterial cells without actual endocytotic uptake.

Cytomorphological changes in liver cells exposed to allyl and benzyl isothiocyanate and their cysteine and glutathione conjugates.

Since allyl isothiocyanate has been reported to be a bladder carcinogen and benzyl isothiocyanate is a known anti-carcinogen, it is important to know the mode of their cytotoxic action. This was investigated in a RL-4 hepatocyte cell line by studying the morphological effects of increasing concentrations of the isothiocyanates and their glutathione and cysteine conjugates. These effects were compared with those induced by tert-butylhydroperoxide which supposedly has its primary effect upon the cytosolic glutathione status and thus upon the integrity of Ca2+-sequestrating mitochondria. The results agree with the previously postulated role of conjugation in the exposure of cells to isothiocyanates: Conjugates show effects similar to those produced by the free parent compounds because conjugates release free isothiocyanates in aqueous solution. The cytomorphological effects increase in a more or less dose-dependent manner with increasing concentrations of isothiocyanate or exposure time. Probably due to increased exposure, suspended RL-4 cells are more sensitive to the toxic action than cells growing on a substrate. No qualitative differences were found between the effects of allyl and benzyl isothiocyanate, indicating that their different effects in vivo are perhaps related to organ-specific differences in equilibrium between the conjugated and unconjugated forms of the test substances. The first cytomorphological effects of isothiocyanates consist of surface blebbing (zeiosis) and swelling of dictyosomal cisternae. At higher concentrations swelling extends to vesicles of endoplasmic reticulum. Mitochondria are not affected until the cells reach the necrotic phase of injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione- and cysteine-mediated cytotoxicity of allyl and benzyl isothiocyanate.

Ally isothiocyanate has been reported to be a bladder carcinogen in male rats. On the other hand, benzyl isothiocyanate is an anti-carcinogen. These contradicting properties led us to investigate the cytotoxicity of these compounds in RL-4 rat hepatocytes. Since conjugation with glutathione plays an important role in the metabolism of these isothiocyanates, the glutathione and L-cysteine derivatives were also tested for cytotoxicity (electron microscopy, trypan blue exclusion, cell attachment, and inhibition of cell division). Both types of conjugates caused considerable toxicity: allyl isothiocyanate conjugates gave effects comparable to the parent compound, but benzyl isothiocyanate was more toxic than its conjugates. Addition of excess glutathione (greater than 4mM) to the free isothiocyanates as well as their conjugates abolished cytotoxicity up to the highest concentration tested (250 microM). Addition of excess L-cysteine (5 to 20 mM) lowered the effects but did not abolish them. The reaction of thiols with isothiocyanates was readily reversible: 15 min after dissolving the conjugates in buffer, pH 7.4, an equilibrium was established in which 9 to 15% of the conjugates was converted to free isothiocyanate. Two hours after addition of 1 mM of the L-cysteine conjugates to medium containing 5 mM glutathione, 80% of the total conjugates was present as the glutathione derivatives. The glutathione conjugates were similarly converted to L-cysteine conjugates. Glutathione conjugates are not able to enter the cell, thus their toxicity is presumably due to the release of free isothiocyanate, and in the presence of excess glutathione no toxicity was observed. L-cysteine derivatives are able to cross the cell membrane, thus excess L-cysteine diminishes cytotoxicity, since less free isothiocyanate is present outside the cells, but does not completely protect the cells. Glutathione and cysteine can be regarded as transporting agents for the isothiocyanates through the body. Initial detoxification can be followed by release of the reactive compound at some other site.