Rudolf I. Salganik

The Benefits and Hazards of Antioxidants: Controlling Apoptosis and Other Protective Mechanisms in Cancer Patients and the Human Population

Cellular oxidants, called reactive oxygen species (ROS), are constantly produced in animal and human cells. Excessive ROS can induce oxidative damage in cell constituents and promote a number of degenerative diseases and aging. Cellular antioxidants protect against the damaging effects of ROS. However, in moderate concentrations, ROS are necessary for a number of protective reactions. Thus, ROS are essential mediators of antimicrobial phagocytosis, detoxification reactions carried out by the cytochrome P-450 complex, and apoptosis which eliminates cancerous and other life-threatening cells. Excessive antioxidants could dangerously interfere with these protective functions, while temporary depletion of antioxidants can enhance anti-cancer effects of apoptosis. Experimental data are presented supporting these notions. The human population is heterogeneous regarding ROS levels. Intake of exogenous antioxidants (vitamins E, C, beta-carotene and others) could protect against cancer and other degenerative diseases in people with innate or acquired high levels of ROS. However, abundant antioxidants might suppress these protective functions, particularly in people with a low innate baseline level of ROS. Screening human populations for ROS levels could help identify groups with a high level of ROS that are at a risk of developing cancer and other degenerative diseases. It also could identify groups with a low level of ROS that are at a risk of down-regulating ROS-dependent anti-cancer and other protective reactions. Screening populations could provide a scientifically grounded application of antioxidant supplements, which could significantly contribute to the nation’s health.

Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes

The mechanism of induction of apoptosis by the novel anti‐cancer drug 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3) was investigated in p53‐defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 μM ET‐18‐OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET‐18‐OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET‐18‐OCH3 in the presence of the antioxidant α‐to‐copherol. Similar results were achieved when cells were switched acutely to choline‐deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET‐18‐OCH3 and CD decreased the phos‐phatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5′,6,6′‐tetramethylbenzimidazolcarbocyanine iodide (JC‐1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the in‐tracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET‐18‐OCH3 or CD. Taken together, these results suggest that the form of p53‐independent apoptosis induced by ET‐18‐OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.— Vrablic, A. S., Albright, C. D., Craciunescu, C. N., Salganik, R. I., Zeisel, S. H. Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine in p53‐defective hepatocytes. FASEB J. 15, 1739—1744 (2001)

Choline deficiency induces apoptosis in SV40-immortalized CWSV-1 rat hepatocytes in culture.

Immortalized CWSV‐1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 μM choline (67‐73% of cells) than when grown in 70 μM choline (2‐3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 μM and 0 μM choline for 72 h. Cells treated with 0 or 5 μM choline for 72 h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55‐75% of cells; 67‐73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24 h, cells grown in 0 or 5 μM choline synthesized DNA more rapidly than those grown in 70 μM choline. By 72 h, the cells grown in 0 or 5 μM choline were forming DNA much more slowly than control cells (assessed by thymidine incorporation, PCNA expression, and mitotic index). Western blot analysis showed that p53 in the nucleus of cells was detected in direct association with SV40 T‐antigen, and was therefore likely to be inactive. We conclude that choline deficiency kills CWSV‐1 hepatocytes in culture by inducing apoptosis via what may be a p53‐independent process, and that this process begins in viable cells before they detach from the culture dish.—Albright, C. D., Liu, R., Bethea, T. C., da Costa, K.‐A., Salganik, R. I., Zeisel, S. H. Choline deficiency induces apoptosis in SV40‐im‐ mortalized CWSV‐1 rat hepatocytes in culture. FASEBJ. 10,510‐516(1996)

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-β1 in immortalized rat hepatocytes†

Transforming growth factor‐β1 (TGFβ1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40‐transformed CWSV‐1 rat hepatocytes that are p53‐defective undergo apoptosis in response to choline deficiency (CD) or TGFβ1, which mediates CD‐apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFβ1 is accompanied by ROS generation and the ROS‐trapping agent N‐acetylcysteine (NAC) inhibits TGFβ1‐induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFβ1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFβ1‐apoptosis. Here we show that CWSV‐1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFβ1‐induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFβ1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFβ1‐induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFβ1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFβ1‐induced apoptosis. J. Cell. Biochem. 89: 254–261, 2003.

Diet, apoptosis, and carcinogenesis

It is known that long-term withdrawal of choline from the diet induces hepatocellular carcinomas in animal models in the absence of known carcinogens. We hypothesize that a choline deficient diet (CD) alters the balance of cell growth and cell death in hepatocytes and thus promotes the survival of clones of cells capable of malignant transformation. When grown in CD medium (5 microM or 0 microM choline) CWSV-1 rat hepatocytes immortalized with SV40 large T-antigen underwent p53-independent apoptosis (terminal dUTP end-labeling of fragmented DNA; laddering of DNA in agarose gel). CWSV-1 cells which were adapted to survive in 5 microM choline acquired resistance to CD-induced apoptosis and were able to form hepatocellular carcinomas in nude mice. These adapted CWSV-1 cells express higher amounts of both the 32 kDa membrane-bound and 6 kDa mature form of TGF alpha compared to cells made acutely CD. Control (70 microM choline) and adapted cells, but not acutely deficient hepatocytes, could be induced to undergo apoptosis by neutralization of secreted TGF alpha. Protein tyrosine phosphorylation is known to protect against apoptosis. We found decreased EGF receptor tyrosine phosphorylation in acutely choline deficient CWSV-1 cells. TGF beta 1 is an important growth-regulator in the liver. CWSV-1 cells express TGF beta 1 receptors and this peptide induced cell detachment and death in control and acutely deficient cells. Hepatocytes adapted to survive in low choline were also resistant to TGF beta 1, although TGF beta 1 receptors and protein could be detected in the cytoplasm of these cells. The non-essential nutrient choline is important in maintaining plasma membrane structure and function, and in intracellular signaling. Our results indicate that acute withdrawal of choline induces p53-independent programmed cell death in hepatocytes, whereas cells adapted to survive in low choline are resistant to this form of apoptosis, as well as to cell death induced by TGF beta 1. Our results also suggest that CD may induce alterations (mutations?) in growth factor signaling pathways which may enhance cell survival and malignant transformation.

Choline deficiency induces apoptosis and decreases the number of eosinophilic preneoplastic foci in the liver of OXYS rats.

Choline deficiency (CD) was previously shown to trigger apoptosis in rat hepatocytes in culture and in vivo. In the present study we investigated the effects of short-term withdrawal of choline from the diet on the expression of putative preneoplastic foci in OXYS rats, an inbred strain with an inherited overproduction of free radicals. Animals were fed a defined, choline-sufficient (CS, control) or choline-deficient (CD) diet for 6 weeks. Eosinophilic, glutathione S-transferase (π class) (+) preneoplastic foci were found in histologic sections of control OXYS rat liver. CD caused a 60% decrease in the number of eosinophilic foci per liver section (27.0 ± 6.1 vs. 10.6 ± 4.6 foci/section) compared to CS controls. Apoptotic bodies were detected in 0.18 ± 0.03% of hepatocytes in CD livers compared to 0.05 ± 0.009% of hepatocytes in controls. Cells which exhibited an apoptotic morphology in hematoxylin and eosin-stained sections were TUNEL-positive, confirming the induction of apoptosis. Also in CD animals compared to controls, there was an increased expression of p27Kip1 protein, and a reduction in PCNA nuclear labeling and the number of mitotic figures, consistent with an inhibition of cell proliferation in the livers of CD animals. This study shows that the liver of OXYS rats with an inherited overgeneration of free radicals retains sensitivity to CD, and that this p53-independent trigger of apoptosis can decrease the number of eosinophilic foci in the livers of these animals.

A p53-dependent G1 checkpoint function is not required for induction of apoptosis by acute choline deficiency in immortalized rat hepatocytes in culture

Abstract Apoptosis is an intrinsic cell program that causes unwanted or damaged cells to commit suicide. It is not well appreciated that nutrients can modulate this significant process. Choline is a nutrient that is crucial for the normal function of all cells, and its absence induces apoptosis. We examined whether p53 or p21 Waf/Cip1 were required for induction of apoptosis by choline deficiency in CWSV-1 rat hepatocytes immortalized with SV40 large T antigen. When grown in a defined, serum-free, choline-sufficient (70 μM) medium, CWSV-1 cells failed to undergo apoptosis in response to γ-irradiation, a well-known p53-dependent trigger of apoptosis. However, primary hepatocytes with an intact p53 gene expressed typical morphologic features of apoptosis and underwent G1 cell cycle arrest in response to γ-irradiation. CWSV-1 cells continued to proliferate following γ-irradiation, which is consistent with the loss of the G1 checkpoint response. These cells also failed to undergo apoptosis in response to cisplatin, whereas the p53-competent primary hepatocytes underwent apoptosis in response to this drug. When maintained for 48 hours in choline-deficient (CD) medium (5 μM choline), CWSV-1 cells exhibited terminal dUTP nucleotide DNA end-labeling assay (TUNEL)-positivity, a DNA ladder typical of internucleosomal DNA fragmentation, and classical cellular features of apoptosis. CD also induced apoptosis in Hep3B hepatocytes, a p53 deletion-mutant. Thus, it is reasonable to suggest that choline deficiency is capable of overcoming a block in the p53 pathway, activating an alternative apoptosis signaling pathway.