Glucuronic acid and sulphate ester conjugation in human lung cancer cell lines.

In short-term organ cultures of normal human peripheral lung, phenolic substrates are metabolized predominantly to sulphate ester conjugates with little or no glucuronic acid conjugates being formed (Mehta & Cohen, 1979). However, with shortterm organ culture of tumour tissue from the lung of the same patients, the converse appears true (R. Mehta & G. M. Cohen, unpublished work). The rationale for this present study was to determine whether cell lines derived from human lung tumours showed similar differences to our preliminary observations on tumour tissue. Eight cell lines were routinely cultured (Ellison et al., 1976) in 90% Dulbecco’s modified Eagle’s medium, 10% bovine foetal calf serum, kanamycin (80mg/litre) and fungizone (0.1 25 mg/ litre) in plastic tissue-culture flasks, maintained at 37OC in CO,/air (1 :9). After having decanted Dulbecco’s medium from each flask, 3ml of the same medium containing either [ l-*‘Cl I-naphthol ( I O ~ M ) or [ 3H]3-hydroxybenzo[alpyrene (2pM) were added to monolayer cell cultures and incubated at 37OC for 90min. The medium was then decanted from the cells and stored at -2OOC until analysis. The conjugates of 1-naphthol in the media were analysed by t.1.c. as previously described (Mehta & Cohen, 1979). Conjugates of 3-hydroxybenzolalpyrene were analysed in a similar way, but were applied to a different silica-gel t.1.c. plate (200mm x 40mm; Kieselgel 60, 0.2 mm thickness; aluminium-backed plates without fluorescent indicator from E. Merck, Darmstadt, Germany). A portion of the culture medium was chromatographed as a 1 : 1 mixture with methanol/acetone (4 : 1, v/v). Small amounts of unlabelled substrate and its glucuronic acid and sulphate ester conjugates were added as unlabelled carriers to this mixture before application to the plate. Chromatograms were developed at room temperature with a solvent system of propan-Iol/NH,/toluene (7 :3 : I , by vol.). Protein determinations were carried out on lysed-cell suspensions by the Lowry technique. The results obtained from the human tumour lung cell lines may be summarized as follows. Overall, I-naphthol appeared to be a much better substrate for metabolism than 3-hydroxybenzo[alpyrene. The glucuronide conjugate of 1 -naphthol had been formed in four of the cell lines, two of which produced this metabolite extensively and exclusively. Of the four remaining cell lines, three virtually did not metabolize the substrate and the fourth, Dre, produced small amounts of sulphate only. Minor quantities of conjugates from 3-hydroxybenzo[alpyrene were produced, although it could be argued that amounts are so small as to mean the majority of the cell lines did not metabolize the substrate. However, the Ben cell line did produce moderate quantities of the glucuronide conjugate.