Copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPX, both Se-dependent and Se-independent), and glutathione reductase (GR) were measured in normal, nitrosoguanidine-transformed and SV40-transformed mouse liver cells in culture, as well as in mouse liver homogenates. Enzyme activities were compared on the basis of 3 different endpoints: per mg protein, per mg DNA, and per 10(6) cells. Except for GR, activity of all the measured anti-oxidant enzymes was much higher in vivo than in vitro. All of the anti-oxidant enzyme activities were lower in general in the 2 transformed cell lines than in the in vitro normal cell line, except Cu-ZnSOD, which showed little change. However, MnSOD was the only enzyme which showed lowered activity in both transformed cell lines, no matter what endpoint was used. This finding is in agreement with previous work showing lowered MnSOD activity in tumor cells.

Vitamin A (retinol) and retinoic acid are necessary for the maintenance of the female reproductive system of higher animals. Our previous work has demonstrated cell specific expression of cellular retinoic acid-binding protein (CRABP) and cellular retinoic-acid binding protein(II) [CRABP(II)] in the uterus of the rat. CRABP(II) expression was shown to be induced in the uterine surface epithelial cells by treatment of prepubertal rats with pregnant mare serum gonadotropin (PMSG). Here we report that, after PMSG treatment, collected uteri had markedly higher levels of retinoic acid than did the uteri of prepubertal rats treated with the control vehicle. Smooth muscle, stromal, and epithelial cells were then cultured from uteri from such animals and provided with retinol or with the retinol/retinol-binding protein complex. Retinoic acid production, analyzed by high-performance liquid chromatography, was observed for the epithelial cells from the uteri of prepubertal animals treated with PMSG, cells previously shown to express CRABP(II) and confirmed here to continue to express it in culture. Little or no retinoic acid was produced by cultured epithelial cells from the prepubertal uteri [shown previously to be negative for CRABP(II)] or by smooth muscle and stromal cells taken from uteri of prepubertal or PMSG-treated rats (shown previously to express CRABP). Retinoic acid production by uterine epithelial cells [and CRABP(II) expression] was also observed if the prepubertal rat was treated with estrogen before cell collection. At no time did cells expressing CRABP exhibit significant retinoic acid synthesis. Thus, this system revealed an important difference in retinoid metabolism between cells expressing CRABP and CRABP(II) and suggests CRABP(II) may participate in retinoic acid production and/or secretion.

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Normal embryonal mouse liver cells in culture were shown to undergo spontaneous transformation during prolonged subculture. The spontaneously transformed cells lost their anchorage dependence, as measured by a soft agar assay, and gave rise to tumors in nude mice. Accompanying this transformation, the antioxidant enzymes, copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT) and glutathione reductase, decreased significantly in activity; the decline in enzymatic activity of CuZnSOD, MnSOD and CAT was due to a decline in the levels of immunoreactive protein. These spontaneously transformed high passage in vitro liver cells appeared similar in morphology, antioxidant enzyme activity and tumorigenicity to their counterparts transformed by N-methyl-N-nitro-N-nitrosoguanidine and Simian virus 40. These data provide experimental evidence that changes in antioxidant enzymes are associated with spontaneous in vitro cellular transformation of mouse embryonal liver cells.

The induction of hsp-70 by gamma irradiation was studied in Chinese hamster ovary (CHO) cells. Synthesis of hsp-70 was assessed by SDS-PAGE of radiolabeled proteins while levels of hsp-70 mRNA were estimated by Northern blot analysis of total RNA. SDS-PAGE analysis indicated that the synthesis of hsp-70 was enhanced in cells exposed to 400 or 1000 Gy. Northern blot analysis of RNA from cells exposed to 1000 Gy indicated a rapid increase in mRNA capable of hybridizing to an hsp-70 oligonucleotide probe. These experiments represent the first report demonstrating that ionizing radiation can increase hsp-70 mRNA.