Regina Schlepegrell

Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells

The data concerning the mutagenic, clastogenic and carcinogenic properties of inorganic lead compounds have been conflicting. To investigate whether the genotoxicity of lead is due to indirect effects such as interference with DNA-repair processes, the induction of mutations, sister-chromatid exchanges and strand breaks by lead ions alone as well as in combination with UV light as a standard mutagen were determined. Lead acetate alone does not induce DNA-strand breaks in HeLa cells or mutations at the HPRT locus and sister-chromatid exchanges in V79 Chinese hamster cells. However, at all endpoints tested, lead ions interfere with the processing of UV-induced DNA damage. They inhibit the closing of DNA-strand breaks after UV irradiation and enhance the number of UV-induced mutations and sister-chromatid exchanges, indicating an inhibition of DNA repair. These data point out the necessity to consider such indirect effects when assessing the genotoxicity of metal compounds. As possible mechanisms of repair inhibition we suggest either the interaction with repair enzymes such as polymerase or ligase or else the interaction with calcium-regulated processes, for example with calmodulin.

Sensitive analysis of oxidative DNA damage in mammalian cells : use of the bacterial Fpg protein in combination with alkaline unwinding

The measurement of oxidative DNA base modifications by different methods has received special attention in recent years. Here we describe a procedure to quantify DNA lesions recognized by the bacterial formamido-pyrimidine-DNA glycosylases (Fpg protein). These include 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) as well as some other forms of imidazole ring-opened purines, which are converted into abasic sites and subsequently into DNA single-strand breaks by the associated endonuclease activity. The frequency of DNA strand breaks is determined by the alkaline unwinding technique. The procedure provides a fast and sensitive tool to assess the extent of spontaneous as well as induced oxidative DNA damage in mammalian cells.

Modulation by Co(II) of UV-induced DNA-repair, mutagenesis and sister-chromatid exchanges in mammalian cells

In bacterial test systems, Co(II) has been shown to be antimutagenic in combination with several chemical and physical agents. To investigate whether such modulations also apply to mammalian cells, the effect of Co(II) on UV-induced mutagenesis, sister-chromatid exchanges as well as DNA damage and its removal was determined. Co(II) itself is weakly mutagenic at the HPRT locus and increases the frequency of sister-chromatid exchanges. Additionally, at both endpoints the metal ions enhance the genotoxicity of UV light. To discriminate between an enhancement of DNA damage and an interference with repair processes, the number of pyrimidine cyclobutane dimers was determined by HPLC. While the induction of these DNA lesions is not affected by Co(II), their removal is inhibited at concentrations of 75 microM Co(II) and higher. Analysis of the kinetics of strand-break induction and closure after UV irradiation by nucleoid sedimentation reveals an accumulation of strand breaks in the presence of Co(II). This indicates that either the polymerization or the ligation step in excision repair is affected. Since similar interactions with the processing of UV-induced DNA damage have been observed with other carcinogenic and/or mutagenic metal ions, this appears to be a common mechanism of metal genotoxicity.

Nickel(II) inhibits the repair of O6-methylguanine in mammalian cells.

Abstract Nickel compounds are widespread carcinogens, and although only weakly mutagenic, interfere with nucleotide excision repair and with the repair of oxidative DNA base modifications. In the present study we investigated the effect of nickel(II) on the induction and repair of O6-methylguanine and N7-methylguanine after treatment with N-methyl-N-nitrosourea (MNU). We applied Chinese hamster ovary cells stably transfected with human O6-methylguanine-DNA methyltransferase (MGMT) cDNA (CHO-AT), and compared the results with the MGMT-deficient parental cell line. As determined by high-performance liquid chromatography/electrochemical detection (HPLC/ECD), there was a slight but mostly not significant reduction in the formation of both types of DNA lesions by MNU in the presence of nickel(II). Although nickel(II) did not markedly affect the repair of N7-methylguanine, it decreased the repair of O6-methylguanine in a dose-dependent manner, starting at concentrations as low as 50 μM. While the MGMT protein level was not altered in the presence of nickel(II), the MGMT activity was diminished as demonstrated in cell extracts form nickel-treated cells. This repair inhibition was accompanied by an increase in MNU-induced cytotoxicity in nickel-treated CHO-AT cells but not in MGMT-deficient control cells. There is strong evidence that O6-methylguanine is involved in tumour formation after exposure to alkylating agents. Thus, the finding that nickel(II) inhibits the repair of this lesion could be of major importance for risk assessment in case of combined exposures at work places and in the general environment.

Uptake and genotoxicity of micromolar concentrations of cobalt chloride in mammalian cells

Literature data concerning the genotoxicity of cobalt salts have been conflicting. To establish appropriate incubation conditions, we conducted a series of uptake studies, before genotoxicity was determined by DNA strand break induction in HeLa cells and mutagenicity in V79 Chinese hamster cells. Co(II) is taken up by HeLa cells in a concentration‐dependent manner and is accumulated inside the cell. The uptake is preceded by a fast association step to the outer membrane, with no saturation up to 24 h. DNA strand breaks as determined by nucleoid sedimentation are induced at concentrations as low as 50μMCoCl2. The induction is time‐dependent, showing the highest number of breaks after 4h incubation with no further increase up to 24h. CoCl2 is mutagenic at the HPRT‐locus, enhancing the spontaneous mutation frequency 4.2‐fold at 100μ?. Besides direct interactions with DNA, the mutagenicity of CoCl2 could also be due to a decrease in the Fidelity of DNA polymerisation.

Genotoxicity of Fe(III)-NTA and its Interaction with other Carcinogenic Metal Compounds

As an essential element in humans, iron is involved in many biochemical reactions; however, it is also known to catalyse the production of highly reactive oxygen species like hydroxyl radicals and superoxide anions, which produce DNA damage in cell free systems (Inoue and Kawanishi, 1987). Since in intact cells iron is mainly bound to storage proteins like ferritin and cellular defense systems exist to detoxify free oxygen radicals, toxic reactions in vivo are prevented to a high degree (for review see Halliwell and Gutteridge, 1984).

Lead(II) Interferes with the Repair and Processing of UV-Induced DNA Damage

Inorganic lead compounds are classified as possibly carcinogenic to humans (International Agency for Research on Cancer, IARC, 1987). Even though the data from epidemiological studies of lead-exposed workers with respect to carcinogenicity are still inadequate, lead has been shown to cause renal tumors and to be cocarcinogenic in experimental animals (reviewed by Swierenga et al., 1987). Regarding in-vitro tests, incorsistent results have been reported about the clastogenicity of inorganic lead compounds (Forni et al., 1980; Gebhart and Rossman, 1989). In a previous report, lead ions were shown to enhance the number of mutants at the HPRT-locus after long-term exposure of V79 cells. However, no direct DNA-damage like strand breaks or DNA-protein crossLinks were detected (Zelikoff et al., 1988). For that reason, we investigated Whether the genotoxicity is rather due to indirect effects like an interference with DNA repair processes, by applying UV as a standard mutagen.