F. Cattabeni

Development and characterization of hydrogen peroxide-resistant Chinese hamster ovary cell variants. I: relationship between catalase activity and the induction/stability of the oxidant-resistant phenotype

Hydrogen peroxide (H2O2)-resistant sublines of Chinese hamster ovary (CHO) cells were isolated by in vitro exposure to the oxidant (treatment for 1 hr followed by 3 days of growth in peroxide-free medium). Stepwise increase in low level H2O2 concentrations produced variants which were progressively more resistant to the growth inhibitory effect elicited by the oxidant. Removal from H2O2 decreased resistance and the curve describing this process was biphasic in nature. In addition, the rate of loss of the H2O2-resistant phenotype was more rapid for the toxicity elicited by low concentrations of hydrogen peroxide, compared to that produced by high concentrations. Changes in total cell proteins were found to parallel the variations in sensitivity to the oxidant, since the protein content constantly increased during the adaptation process and decreases upon removal from H2O2. Catalase activity did not show large variations in resistant sublines with respect to the parental cell line, and these changes were at least partially related to differences in cell size/amount of total cell proteins of the sublines. In addition, the minor changes observed for catalase activity did not correlate with the degree of resistance to growth inhibition elicited by the oxidant. It may therefore be suggested that the H2O2-resistant phenotype of mammalian cells, initially adapted to low--then gradually increased--concentrations of the oxidant, is the result of a complex phenomenon which only partially involves over-expression of catalase.

L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity: relationships between dna single/double strand breakage and cell killing

Results presented in this study demonstrate an association between the L-Histidine-mediated enhancement of H2O2-induced cytotoxicity and the formation of DNA double strand breakage (DSB), whereas no relationship exists between the increased cytotoxic response and DNA single strand breakage (SSB). Indeed, the higher lethality and the production of DNA DSB occurred in oxidatively-injured cells regardless of whether the exposure to L-Histidine was performed before or during challenge with the oxidant. In fact, the increased level of DNA SSB detected in cells simultaneously exposed to the oxidant and the amino acid was not observed in cells pre-treated with L-Histidine and then challenged with hydrogen peroxide. Further experiments have demonstrated an association between the kinetics of DNA DSB formation and the enhancement of the cytotoxic response. In conclusion, intracellular L-Histidine seems to mediate the formation of DNA DSB and the increased growth-inhibitory response elicited by the oxidant. In addition, these results suggest that the enhancement of DNA SSB is produced by the extracellular/plasma membrane fraction of the amino acid and not causally related to the L-Histidine-mediated increase of the growth-inhibitory response to H2O2-treated cells.

Development and characterization of hydrogen preoxide-resistant Chinese hamster overy (CHO) cell variants—II. Relationships between non-protein sulfydryl levels and the induction/stability of the oxidant-resistant phenotype

Hydrogen peroxide sensitive and resistant sublines of Chinese hamster ovary (CHO) cells were tested for their non-protein sulfhydryl (NPSH) content in an attempt to establish whether a relationship exists between resistance to growth inhibition elicited by the oxidant and the NPSH pool. Cell variants characterized by increasing levels of resistance to hydrogen peroxide displayed a significant increase in cellular NPSH (expressed on a per million cell basis). Growth of resistant cells for various lengths of time in the absence of H2O2 decreased resistance, whereas the NPSH content did not vary (at least up to 127 days of growth in peroxide-free medium). The NPSH pool returned to control levels after an additional 82 days. These changes, however, were probably related to differences in cell size/amount of total cell proteins in the sublines. Indeed, when NPSH levels were expressed on a per milligram protein basis, essentially no variations were observed in sensitive and resistant sublines. It is important to note that, even by expressing the NPSH content on a per million cell basis, no correlation was found with the degree of resistance to growth inhibition elicited by the oxidant. Further experiments have demonstrated that, under conditions of reduced NPSH content (obtained by growing the cells in the presence of a glutamylcysteine synthetase inhibitor), the cytotoxic action of hydrogen peroxide was very slightly, if at all, augmented in both wild type and resistant cells. We may therefore conclude that cellular NPSH do not afford significant protection against growth inhibition induced by hydrogen peroxide in wild type cells, and that the same lack of effect occurs in cells with an increased NPSH content and carrying the oxidant-resistant phenotype.

Regulatory role of extracellular medium components in metal induced cyto- and geno-toxicity.

Recently, a considerable amount of work in different laboratories has been devoted to establishing and characterizing the mutagenicity and the oncogenic potential of metal compounds in cell culture systems. Monolayer cultures offer several advantages that make their use attractive for short term studies. However, the entry of metals into cells can be affected by various types of interactions occurring between metal compounds and medium components. Different types of sera (newborn vs fetal bovine), different amounts or even different batches of serum significantly affect the toxicity of several metal ions such as Ni/sup 2 +/, Cd/sup 2 +/, Hg/sup 2 +/. In this paper the authors have characterized some of these effects and studied cyto- and geno-toxic actions produced by HgCl/sub 2/ or CaCrO/sub 4/ in Chinese Hamster Ovary (CHO) cells grown as monolayer cultures.

Induction of DNA double strand breaks in cultured mammalian cells exposed to hydrogen peroxide and histidine

The amino acid histidine was found to increase the toxicity of H2O2 in cultured mammalian cells. Histidine also augmented the level of DNA single strand breaks (SSB) detectable in cells exposed to the oxidant and, in addition, resulted in the appearance of DNA double strand breaks (DSB), a lesion which is not produced by H2O2 alone.

The L-histidine-mediated enhancement of hydrogen peroxide-induced DNA double strand breakage and cytotoxicity does not involve metabolic processes

The cytotoxic response of Chinese hamster ovary (CHO) cells to challenge with hydrogen peroxide was highly dependent upon the temperature of exposure, being markedly higher at 37 degrees than at 4 degrees C. Increasing intracellular levels of L-histidine prior to challenge with hydrogen peroxide increased the toxicity elicited by the oxidant at both physiologic and ice-bath temperatures. The effect of the amino acid, however, was more pronounced under conditions at 4 degrees C, as compared to 37 degrees C. Indeed, at 4 degrees C the oxidant was nontoxic at submillimolar levels and pre-exposure to L-histidine restored cytotoxicity to levels slightly higher than those observed after treatment at 37 degrees C (in the micromolar range). Pre-exposure to the amino acid increased the production of DNA double-strand breaks (DSBs) elicited by treatment with the oxidant both at 37 degrees and 4 degrees C. A remarkable correlation was found when the level of this lesion was plotted against the cytotoxic response observed using different concentrations of L-histidine or hydrogen peroxide, or treating the cells with the oxidant either at 37 degrees or 4 degrees C, thus suggesting the existence of a cause-effect relationship. The overlapping correlation curves obtained with cells challenged with the oxidant at 4 degrees or 37 degrees C also suggest that similar molecular mechanisms mediate the formation of DNA DSBs under both experimental conditions. Two lines of evidence provide experimental support for this inference: (1) the kinetics of repair of DNA DSBs generated at 37 degrees or 4 degrees C were virtually superimposable; this would suggest that the same repair pathway(s) is/are responsible for the removal of DNA DSBs generated at the two temperatures; and (2) the size distribution of double-stranded DNA fragments produced under the two treatment conditions, resulting in a similar cytotoxic response, was basically identical. This is indicative of remarkable similarities in the topology of chromosomal domains where DSBs are generated. Overall, the results presented in this paper provide further experimental evidence supporting the notion that DNA DSBs are responsible for the L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity, and demonstrate that the mechanism whereby the amino acid enhances the ability of hydrogen peroxide to produce DNA double strand breakage and cell killing does not depend on cellular metabolism and/or energy-dependent reactions.

Evidence for a dissimilar mechanism of enhancement of inorganic or organic hydroperoxide cytotoxicity by L-histidine

L-Histidine markedly increases inorganic and organic hydroperoxide-induced cytotoxicity and DNA single-strand breaks (SSBs) in Chinese hamster ovary cells. These effects were prevented by the iron chelator o-phenanthroline and were insensitive to the antioxidant N,N-diphenyl-1,4-phenylenediamine. An excess of L-glutamine, a competitive inhibitor of L-histidine uptake, prevented the L-histidine-mediated enhancement of cytotoxicity induced by both inorganic and organic peroxides. L-Glutamine did not affect the level of DNA SSBs produced by H2O2/L-histidine, although it abolished the enhancement of SSB formation triggered by L-histidine in cells exposed to the organic peroxides. DNA SSBs generated by the organic hydroperoxides either alone or associated with L-histidine were removed with superimposable kinetics, whereas those produced by H2O2 in the presence of the amino acid were repaired more slowly than SSBs produced by the oxidant alone. DNA double-strand breaks, which are considered to be highly cytotoxic, were detected only in cells treated with H2O2 and L-histidine. Finally, L-histidine was shown to markedly increase the extent of mitochondrial damage produced by organic but not by inorganic hydroperoxides.

Modulation of the oxidative response of cultured mammalian cells by L-histidine.

Previous studies have demonstrated that L-histidine increases the fragmentation of purified DNA induced by hydrogen per~xide l -~ as well as other effects produced by the oxidant in cultured mammalian cells, such as cytotoxicity,J chromosomal aberrations: sister chromatid exchange~ ,~J~~ and the formation of r n i c r o n u ~ l e i . ~ ~ ~ ~ ~ The mechanism(s) involved in the enhancing effect of L-histidine on the deleterious effects of hydrogen peroxide is (are) still controversial, although various hypotheses have been made.Summarized here are some of our recent results in studies to investigate the effects of L-histidine on DNA damage produced by hydrogen peroxide in cultured mammalian cells. L-Histidine (over a range of 0.1-1 mM) markedly and dose dependently increases the inhibitory effects on growth and DNA synthesis elicited by hydrogen peroxide in cultured Chinese hamster ovary (CHO) cells. In agreement with previously published data,7 DNA single-strand breakage was also higher (about two times) in the presence of the amino acid. Under experimental conditions in which similar levels of initial damage were produced, with the oxidant alone or associated with L-histidine, we found that these latter breaks were characterized by a slower rate of repair (tl,z of 40 minutes) than were breaks generated by the oxidant alone (tl/Z of 15 minutes). In the presence of L-histidine, hydrogen peroxide also produced DNA double-strand breakage, a lesion that cannot be detected in cells treated even with exceedingly high concentrations of the oxidant alone. Enhancement of Hz02-induced cytotoxicity mediated by L-histidine is always associated with the formation of DNA doublestrand breakage. This relation does not exist for DNA single-strand breaks. Indeed, the increased cytotoxic response and the production of DNA double-strand breaks were apparent and similar in cells in which exposure to L-histidine was performed before or during challenge with the oxidant. On the contrary, the increased level of DNA single-strand breaks detected in cells simultaneously exposed to the oxidant and the amino acid was not observed in cells pretreated with L-histidine and then challenged with hydrogen peroxide. L-Glutamine, an amino acid that shares with L-histidine the same transport system through the plasma membrane, inhibited, in a concentration-dependent fashion, the L-histidine-mediated enhancement of HzOz-induced cytotoxicity. L-Glutamine also prevented the induction of DNA double-strand breaks, but it did not