Antioxidant enzyme levels as a function of growth state in cell culture.

Manganese superoxide dismutase (MnSOD) levels were monitored as a function of time in culture to determine whether these levels were altered at logarithmic growth versus when the cells exhibited density limitation of growth. For comparison, activities of the antioxidant enzymes copper, zinc superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase were also evaluated. Four cell lines were studied, two of which exhibited density limitation of growth and two of which did not. Each cell line showed a unique antioxidant enzyme profile. The two cell lines that showed density limitation of growth also demonstrated induction of MnSOD at the time when the cells stopped proliferating in culture, whereas the other two cell lines did not show induction of MnSOD. There was no strict correlation between density limitation of growth and activities of the other antioxidant enzymes. To determine whether SOD varied with various phases of the cell cycle, NIH/3T3 cells were synchronized using serum starvation, and then SOD activities were measured during quiescence (G0) and the phase of DNA synthesis (S-phase). MnSOD was decreased during S-phase compared with G0, whereas CuZnSOD was increased during S-phase compared with G0, demonstrating alteration of SOD activities with varying phases of the cell cycle. This study suggests the possibility that increased MnSOD may correlate with decreased cell proliferation and suggests significant alterations in SOD activities during the cell cycle.

In vitro modulation of antioxidant enzyme levels in normal hamster kidney and estrogen-induced hamster kidney tumor.

Antioxidant enzyme (AE) activities were studied in normal hamster kidney proximal tubules and in estrogen-induced hamster kidney cancer. In vivo, kidney tumor had lower activities of manganese superoxide dismutase (MnSOD), copper, zinc superoxide dismutase, catalase, and glutathione peroxidase than kidney proximal tubules. Differences in AE activities were, in general, maintained in tissue culture, with AE activities remaining low in tumor cells compared to normal cells. Normal proximal tubular cells showed significant induction of MnSOD activity as a function of time in culture or following exposure to diethylstilbestrol, a synthetic estrogen, while MnSOD activity remained low in tumor cells under these conditions. Our results suggest that antioxidant enzymes, particularly MnSOD, are regulated differently in estrogen-induced hamster kidney tumor cells than in normal kidney proximal tubular cells, demonstrating that cancers arising from hormonal influence have similar AE profiles to those previously described in cancers arising from viral or chemical etiologies.

Laryngeal movement, oropharyngeal pressure, and submental muscle contraction during swallowing.

This study examined temporal relationships of laryngeal movement, oropharyngeal pressure and submental muscle contraction during swallowing. Techniques used to obtain the temporal measures were electroglottography (EGG), oropharyngeal manometry, and submental surface electromyography. Liquid bolus swallows were performed by 40 normal subjects evenly divided by young and elderly, men and women. Results of this investigation suggest that the EGG waveform is reflective of the temporal aspects of laryngeal movement during swallowing and that the EGG has potential as a behavioral modification technique in swallowing therapy. A case study is presented to illustrate the use of the electroglottograph for biofeedback.

In vitro characterization of estrogen induced Syrian hamster renal tumors: comparison with an immortalized cell line derived from diethylstilbestrol-treated adult hamster kidney.

Primary diethylstilbestrol-induced kidney tumors from Syrian hamsters were grown in vitro and maintained in culture for 6 mo. Combined immunohistochemical studies using antibodies to intermediate filaments and ultrastructural studies of tumor cells in culture exhibited characteristics similar to tumor cells in vivo. Furthermore, the cells manifested transformed properties in culture; they grew both as multilayered colonies attached to the tissue culture substrate and as floating multicellular colonies (spheroids). When cultured cells were injected into diethylstilbestrol-treated recipient hamsters, tumors developed at the injection sites. In contrast, renal tubules or whole kidney cortex from control hamsters cultured in the same medium underwent only short-term growth, with senescence developing after approximately 1 mo. However, cell cultures of kidney cortex from animals treated in vivo for 5 mo. with diethylstilbestrol formed a cell line. This diethylstilbestrol-induced cell line has been maintained in culture for 1.5 yr and has the following characteristics: a) it is anchorage-dependent, b) it is negative in in vivo tumorigenicity tests, and c) cultured cells are histochemically and ultrastructurally similar to cultured tumor cells. This culture system should prove to be of use in studying hormonal carcinogenesis in vitro.

Antioxidant enzymes and steroid-induced proliferation of kidney tubular cells.

Diethylstilbestrol induces proliferation of Syrian hamster renal proximal tubular cells. By counting the number of cells in culture, we showed that liposomes containing superoxide dismutase or catalase suppressed diethylstilbestrol-induced proliferation, whereas empty liposomes or liposomes containing inactivated superoxide dismutase did not. Liposomes containing antioxidant enzymes did not suppress proliferation of cells in control media or of cells treated with ethinyl estradiol. In the absence of liposomes, exogenous superoxide dismutase did not suppress diethylstilbestrol-induced proliferation. The decrease in cell number when diethylstilbestrol-treated cells were treated with antioxidant enzyme-containing liposomes was not due to decreased cell viability. Results were confirmed by measuring a correlate of cell proliferation immunohistochemically, using an antibody to proliferating cell nuclear antigen. A larger proportion of diethylstilbestrol-treated cells than of control cells showed nuclear immunostaining with this antibody. The number of cells immunostained in diethylstilbestrol-treated cultures was sharply decreased by the addition of superoxide dismutase- or catalase-containing liposomes. Our studies suggest a role for active oxygen species in diethylstilbestrol-induced proliferation of cultured proximal tubular cells.