Synergistic DNA damage by oxidative stress (induced by H2O2) and nongenotoxic environmental chemicals in human fibroblasts.

Genotoxic combination effects of oxidative stress (induced by H2O2) and eight nongenotoxic environmental chemicals (4-chloroaniline, 2,3,4,6-tetrachlorophenol, lindane, 2,4-dichloroacetic acid (2,4-D), m-xylene, glyphosate, nitrilotriacetic acid and n-hexanol) were determined in human fibroblasts. Genotoxicity was measured quantitatively by the single cell gel electrophoresis assay. The nongenotoxic chemicals were used in non cytotoxic concentrations. H2O2 was used in concentrations producing low (50 microM) and no cytotoxicity (40 microM). All environmental chemicals acted in a synergistic way with H2O2 except DMSO which effectively inhibited H2O(2)-induced DNA damage. The most effective enhancers were 4-chloroaniline, 2,3,4,6-tetrachlorophenol, m-xylene, and n-hexanol. Synergistic effects of hexanol/H2O2 were still evident at a concentration of 0.09 noec (no observed effect concentration). In contrast to synergistic DNA damage in the cell antagonism was found measuring DNA breakage in isolated PM2 DNA. From the results we concluded that synergisms between H2O2 and nongenotoxic chemicals may be a general phenomenon which is not observed on the level of isolated DNA.

Synergistic DNA damaging effects of 4-nitroquinoline-1-oxide and non-effective concentrations of methyl methanesulfonate in human fibroblasts.

DNA damage and DNA repair in human fibroblasts induced by the combination mixture of the genotoxic agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) were studied using the comet assay and the unscheduled DNA synthesis (UDS), respectively. Cells were simultaneously treated for 1h with the no observed effect concentration (noec) of MMS and increasing concentrations of 4-NQO or vice versa. Different results were obtained with the two types of mixtures. When the noec of 4-NQO was combined with increasing concentrations of MMS, no combination effects were observed. However, in experiments with increasing concentrations of 4-NQO and the noec of MMS, an increase in DNA damage and repair (and an enhancement of cytotoxicity) was demonstrated. Quantitative analysis of the effects by the isobologram method confirmed synergistic responses in both tests. We are proposing interactive actions between 4-NQO and MMS, whereby 4-NQO facilitates the attack of MMS on the DNA bases.

Suitability of different cytotoxicity assays for screening combination effects of environmental chemicals in human fibroblasts.

Cytotoxicity screening assays measuring survival, growth, colony forming ability, DNA and protein synthesis in human fibroblasts were tested for their suitability to determine combination effects. Thereby, the dose-response curves of a hydrophilic substance A alone and after pretreatment with a membrane damaging substance B were compared. Substances B were applied at concentrations which did not induce toxic effects in the assays (noec). Synergistic combination effects were demonstrated by reduction of the EC20 value of substances A in the combination in comparison to substances A alone. The following substance pairs (substance B/substance A = membrane damaging/hydrophilic) were tested: n-dodecylbenzenesulfonic acid/2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol/2,4-dichlorophenoxyacetic acid, 1,1,2,2-tetrachloroethane/4-chloroaniline, pentachlorophenol/CrCl3. While survival, growth, and DNA synthesis assays were suitable methods for detecting synergistic combination effects, the growth assay was the most sensitive. Here, all four substance pairs showed synergistic combination effects.

Correlation of synergistic cytotoxic effects of environmental chemicals in human fibroblasts with their lipophilicity.

The cytotoxic combination effects of 2,4-D with 12 xenobiotics having different lipophilicity were investigated in human fibroblasts at their no effect concentrations (NOEC). Each of the chemicals tested in binary combinations enhanced the toxicity of 2,4-D. These synergistic combination effects were independent of the chemical structure of the test compounds. However, the NOEC's of the xenobiotics used in the combinations varied by a factor of 10,000. For strongly lipophilic compounds the lowest NOEC's were needed to induce synergistic cytotoxicity. A linear regression analysis of the concentrations (NOEC's) of the 12 combined xenobiotics against their lipophilicity revealed a correlation with r = 0.96 for 11 agents. This close correlation may be explained by the membrane damaging properties of lipophilic compounds which enhance the uptake of hydrophilic agents.