Alachlor, metolachlor, and propachlor are widely used chloroacetanilide herbicides. Their cytotoxicity in rat (Fa32) and human (Hep G2) hepatoma-derived cells was investigated, in connection with their influence on the endogenous glutathione (GSH) content, on the xenobiotic-metabolizing phase I enzymes 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD), and phase II glutathione transferase (GST). The cytotoxicity was measured by the neutral red uptake inhibition assay. The following toxicity range was observed in both cell lines: propachlor > alachlor > metolachlor. When the endogenous GSH content was reduced by pretreatment of the cells with L-buthionine (S,R)-sulfoximine, the cytotoxicity of the herbicides increased strongly in both cell lines. EROD and PROD activities were dose-dependently increased to different degrees in Fa32, as was EROD in Hep G2, but no PROD activity was observed in these cells. The GSH content was not altered after 1 h treatment, and was approximately doubled after 24 h. GST activity was increased in Fa32 cells but not in Hep G2. A comparable cytotoxicity was observed for the investigated chloroacetanilides in both the rat and the human cell lines. Different interactions with xenobiotic-metabolizing phase I and II enzymes were observed, and GSH showed a protective effect against the acetanilides in both cell lines.

A previously described neutral red uptake-inhibition assay on cultured fathead minnow (FHM) fish cells revealed a good correlation with fish toxicity data. The assay was made more sensitive by reducing the number of cells, by using a longer treatment period, and by simultaneous treatment of the cells with sodium dodecyl sulphate and L-buthionine (S,R)-sulphoximine. The fluorimetrically quantified protein content was then used as the endpoint. The results were compared with toxicity data obtained by the Microtox® method with the bacterium, Vibrio fischeri. A series of 82 surface-water samples were investigated. Cell quantity-dependent and sample concentration-dependent reductions in total protein content were observed. In all, 32 samples were toxic in the FHM assay. Of the seven samples that were shown to be toxic in the Microtox assay, only three were also toxic in FHM cells. No linear relationship was found for the toxicity results obtained with the two methods. Further research to explain the significance of the many positive responses and to identify possible confounding factors will increase the reliability of the sensitised FHM cell assay.

Glutathione transferase (GST) was purified from the microsomes of rat liver by glutathione affinity chromatography. The interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1,4-benzoquinone with microsomal GST was investigated and compared with cytosolic GST. The kinetic inhibition pattern of 1,4-benzoquinone towards microsomal GST was found to be different from that towards cytosolic GST. Microsomal GST purified by affinity chromatography was inhibited by 2,4-D in a non dose-dependent manner, while the crude microsomal GST was inhibited in a dose-dependent manner. This difference was shown to be induced by a reaction on the affinity column, and not by Triton X-100 (also shown to be a GST inhibitor), glutathione, or the elution buffer 0.2% Triton X-100 and 5 mM glutathione in 50 mM Tris-HCl, pH 9.6. The binding of microsomal GST to the affinity matrix caused a partial inactivation of the active site for 2,4-D interaction. The results show that the properties of soluble GST enzymes may not be extrapolated to the microsomal ones.