Glutathione transferase alpha as a marker for tubular damage after trichloroethylene exposure.

To investigate possible persistent nephrotoxic effects of trichloroethylene (TRI), a retrospective study was carried out on 39 workers exposed to high levels of TRI from 1956 to 1975. Total protein levels in urine, as well as serum and urine creatinine and serum urea were unchanged in comparison with the control. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to differentiate between tubular and/or glomerular dysfunction. Urinary excretion of alpha-1-microglobulin and glutathione transferase (GST) alpha, as markers of proximal tubular damage, were correlated with the SDS-PAGE patterns of urinary proteins both in the TRI exposed and the control group. GST alpha was found in elevated concentrations in the urine of the TRI-exposed workers. No increase of urinary GST alpha was observed in the control group, even when alpha-1-microglobulin was elevated as a result of non-toxic damage. Both in the control and exposed groups, GST pi, a marker of distal tubular damage, was in the normal range. The results show that chronic exposure to high doses of TRI causes persistent changes to the proximal tubular system of the kidney and that GST alpha excretion into the urine is a marker well suited for quantitation of the extent of renal damage.

Pathological excretion patterns of urinary proteins in renal cell cancer patients exposed to trichloroethylene.

A study was carried out to investigate urinary protein excretion patterns by means of SDS-polyacrylamide-gel-electrophoresis (SDS-PAGE) in renal cell cancer patients who had previously been exposed to high levels of trichloroethylene. Thirty-eight out of 41 (93%) renal cell cancer patients investigated had former extensive trichloroethylene exposure, but only 23 out of 50 (46%) renal cell cancer patients without a history of occupational exposure to trichloroethylene revealed urinary protein patterns indicative of toxic effects on the tubular system. One hundred controls without histories of overt renal disease and not occupationally exposed to trichloroethylene were examined in the same way; only 11 (11%) of them displayed protein excretion patterns indicative of damage to the renal tubule. These results are supported by alpha 1-microglobulin excretion data. The following conclusions are drawn: (1) Substantially more cases of tubular damage are found amongst renal cell carcinoma patients having been exposed to substantial levels of trichloroethylene over many years as compared with renal cell carcinoma patients not exposed to trichloroethylene. (2) The results support the view that chronic tubular damage is a precondition for the nephrocarcinogenic effect of trichloroethylene. (3) The findings indicate that urine protein patterns, on the basis of the SDS-PAGE methodology, represent a 'biological effect parameter' for the medical surveillance of persons occupationally exposed to trichloroethylene.

ELISA procedures for the quantitation of glutathione transferases in the urine.

The proximal portion of the human kidney tubular system contains the alpha form, while the distal portion contains the pi form of glutathione transferase. These cytoplasmic proteins are released into the urine under pathological conditions, and an ELISA procedure has been developed for their quantitation. Optimal conditions with respect to concentrations of antibody and antigen and incubation times were determined. The procedure developed can detect as little as 0.5 ng enzyme per ml urine, even in the presence of high concentrations of other proteins. No cross reaction between these two isoenzymes or with a number of other proteins in the urine was observed. Antibodies interacted with these antigens in urine samples in the same manner as they interacted with the purified proteins. Storage of samples without loss of antigen required the presence of low concentrations of detergent, such as Tween 20, which both stabilized the enzymes and prevented their adsorption to the walls of the plastic tubes. The results indicate that increased urinary levels of these two enzyme proteins, as determined by the ELISA procedure, are useful markers for tubular damage.

Urinary pi-class glutathione transferase as an indicator of tubular damage in the human kidney.

Glutathione transferase-pi released from kidney tubular epithelial cells was analyzed in the urine of recipients of renal allografts. Urinary content of alpha-class glutathione transferase was also determined for comparison. Control urine from healthy individuals contained detectable levels of the pi-isoenzyme (6.6 +/- 0.46 ng/ml, mean +/- SEM) and this concentration was not increased in the urine of patients demonstrating cyclosporine A-induced nephrotoxicity (6.3 +/- 0.29 ng/ml), in contrast to the alpha-form. Acute rejection increased excretion of the pi-isoenzyme (19.0 +/- 2.0 ng/ml), but not of the alpha-glutathione transferase. Thus, while the serum creatinine level increases in connection with both cyclosporine A-induced nephrotoxicity and acute rejection, analyses of urinary glutathione transferases distinguish well between these conditions. Acute tubular necrosis and renal transplant infarction resulted in a rapid elevation in urinary levels of both alpha- and pi-transferase. The advantages of this approach are that release of the protein into the urine occurs rapidly after tubular damage, the assay is sensitive and specific and can also distinguish between certain pathological conditions. These studies thus indicate that the urinary level of glutathione transferase-pi can be used for monitoring certain pathological processes in the kidney. Quantitation of this enzyme complements the information obtained by measurement of glutathione transferase-alpha.

Quantitation of glutathione transferase-pi in the urine by radioimmunoassay.

A radioimmunoassay procedure for the quantitation of glutathione transferase-pi was developed in order to determine the levels of this protein in human urine. The enzyme was isolated from human placenta with a purification factor of 366 (compared to the original high-speed supernatant fraction), and upon gel electrophoresis, only a single band was seen. Polyclonal antisera were subsequently raised in rabbits and found to be suitable for a radioimmunoassay. Glutathione transferase-pi was localized immunohistochemically to the cells of the distal tubules, the thin loop of Henle and the collecting ducts in the kidney. In contrast, the alpha-isoenzyme was localized exclusively in the proximal tubular epithelium. Samples of urine from healthy individuals contained about 6 ng of the pi-transferase/ml. The method proved to be specific for glutathione transferase-pi, and no cross-reaction with the alpha- or mu-transferase or with other proteins occasionally appearing in urine occurred. The protein was quite stable upon storage and insensitive to variations in the urine pH. Thus, it appears that glutathione transferase-pi can be conveniently quantitated by radioimmunoassay and changes in the concentration of this protein in human urine thus monitored.

Immunohistochemical localization of alpha and pi class glutathione transferases in normal human tissues.

The distribution of alpha and pi class glutathione transferases in autopsy and biopsy samples of normal human tissues was investigated by immunohistochemistry. The class alpha glutathione transferases exhibited restricted distribution. Intensive staining was visible in all hepatocytes, in kidney proximal tubular cells, in the zona reticularis of adrenal cortex and in Leydig cells of testis. Staining of lesser intensity could also be observed in the gastrointestinal epithelium, exocrine pancreas and some bile and pancreas ducts. In colon and gall bladder only nuclei were stained, but in the other tissues both nuclei and cytoplasm contained alpha class glutathione transferases. Glutathione transferase pi exhibited a more general distribution and could be observed in epithelia of the respiratory, gastrointestinal and urinary tracts, in all endocrine cells investigated, and also in the exocrine glands of prostate, in smooth muscle, adipocytes, blood vessel endothelium and placenta. It was also visible in the Schwann cells of peripheral nerves and in the choroid plexus. In gall bladder and colon only nuclei were stained, while in the intrahepatic bile ducts only cytoplasm was stained. All other positive cells exhibited glutathione transferase pi in both nuclei and cytoplasm.