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.