Modeling the inactivation kinetics of bacillus spores by glutaraldehyde.

AIMS: Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. METHODS AND RESULTS: We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality-controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. CONCLUSIONS: The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time-duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. SIGNIFICANCE AND IMPACT OF THE STUDY: The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde-based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.

Wet and dry density of Bacillus anthracis and other Bacillus species.

AIMS: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1.162 and 1.165 g ml(-1) and the other group included strains whose spores showed higher density values between 1.174 and 1.186 g ml(-1). Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R(2) = 0.95) between density and volume for the spores of all strains and species studied. CONCLUSIONS: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.

Analysis of the survival of Venezuelan equine encephalomyelitis virus and possible viral simulants in liquid suspensions.

AIMS: To compare the inactivation rate of Venezuelan equine encephalomyelitis (VEE) virus in liquids to that of Sindbis virus (SV, another alphavirus) and to a bacteriophage (MS2) generally used as a viral simulant in the development of countermeasures in biodefense. METHODS AND RESULTS: Viruses were inoculated into liquids and viral titres were determined at various times postinoculation. The viruses were stable in distilled-deionized (dd) water at 4 degrees C during the 21 days of the study. The inactivation rates of VEE and SV in dd water at 21 and 30 degrees C were very similar (between 0.12 and 0.14 log(10) per day), while MS2 was three-fold slower. In tap water (chlorine content between 4 and 5 ppm) at 21 degrees C, VEE and SV were inactivated at twice the rate measured in dd water. CONCLUSIONS: The inactivation rates of VEE and SV were similar to each other and faster than MS2 in all liquids tested. SIGNIFICANCE AND IMPACT OF THE STUDY: VEE is likely to remain viable for many days after release into water, snow, or even chlorinated tap water. SV can be used to estimate the persistence of VEE in liquids, but using MS2 as a simulant would overestimate of the stability of VEE.

Difference between the spore sizes of Bacillus anthracis and other Bacillus species.

AIMS: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 microm and 0.86 +/- 0.08 microm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 microm or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 microm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. CONCLUSIONS: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants.

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.

Resistance of Pseudomonas aeruginosa to liquid disinfectants on contaminated surfaces before formation of biofilms.

A comparison was made of the effectiveness of popular disinfectants (Cavicide, Cidexplus, Clorox, Exspor, Lysol, Renalin, and Wavicide) under conditions prescribed for disinfection in the respective product labels on Pseudomonas aeruginosa either in suspension or deposited onto surfaces of metallic or polymeric plastic devices. The testing also included 7 nonformulated germicidal agents (glutaraldehyde, formaldehyde, peracetic acid, hydrogen peroxide, sodium hypochlorite, phenol, and cupric ascorbate) commonly used in disinfection and decontamination. Results showed that P. aeruginosa is on average 300-fold more resistant when present on contaminated surfaces than in suspension. This increase in resistance agrees with results reported in studies of biofilms, but unexpectedly, it precedes biofilm formation. The surface to which bacteria are attached can influence the effectiveness of disinfectants. Viable bacteria attached to devices may require dislodging through more than a one-step method for detection. The data, obtained with a sensitive and quantitative test, suggest that disinfectants are less effective on contaminated surfaces than generally acknowledged.

Cytotoxicity of liquid disinfectants.

This study was prompted by toxic responses to disinfecting agents reported in patients after surgical procedures and in sensitized health care personnel. We evaluated the cytotoxicity of seven substances used in the formulation of common liquid chemical disinfectants and sterilants. We found that a standard method based on direct microscopic examination of cell cultures was insensitive and may result in an underestimation of the risk that disinfectants pose to health care personnel or patients who are exposed to these substances. Using independent quantitative tests measuring the integrity of the cellular membrane, metabolic activity, or cell growth, we found that there is a several-hundredfold difference in the relative toxicity of various disinfecting substances. The concentration toxic in 50% of the cell population (TC(50)) that was found for each disinfectant was similar in a variety of cell lines from human, monkey, or mouse origin. Statistical analysis of TC(50)s suggests that liquid disinfecting agents could be classified in three main groups according to their relative toxicity, with: (1) mild (TC(50) > 1 mM, including phenol, hydrogen peroxide, and formaldehyde); (2) moderate (1mM > TC(50) > 0.1 mM, sodium hypochlorite); and (3) severe (TC(50) < 0.1 mM, glutaraldehyde, cupric ascorbate, and peracetic acid) toxicity. These data suggest a vast difference in the potential risk of various disinfectants and sterilants. The data presented in this study should help to define the relative toxic risk of different disinfecting substances to patients and health care personnel and assist in the selection of safer microbicidal formulations.

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine.

The interaction of chelators and reducing agents is of particular importance in understanding iron-associated pathology since catalytic iron undergoes cyclic reduction and oxidation in vivo. Therefore, we treated plasmid DNA with free or chelated Fe(III) in the presence of biological reductants, and simultaneously measured the number of single strand breaks (SSBs) and oxidative base modification (8-hydroxy-2'-deoxyguanosine; 8-OHdG) by quantitative gel electrophoresis and HPLC with electrochemical detection, respectively. Production of SSBs and 8-OHdG was linearly correlated suggesting that these two different lesions share a common chemical mechanism. The levels of both lesions were enhanced when Fe(III) was chelated to citrate or nitrilotriacetic acid. Reducing agents showed different potency in inducing DNA damage catalyzed by chelated iron (L-ascorbate > L-cysteine > H2O2). Chelation increased SSB formation by approximately 8-fold and 8-OHdG production by approximately 4-fold. The ratio of SSB/8-OHdG catalyzed by chelated iron, which is twice as high as by unchelated iron, indicates that chelation affects iron-catalyzed oxidative DNA damage in a specific way favoring strand breakage over base modification. Since iron is mostly chelated in biological systems, the production of genomic and mitochondrial DNA damage, particularly strand breaks, in diseases involving iron overload is likely to be higher than previously predicted from studies using unchelated iron.

Bacterial spores survive treatment with commercial sterilants and disinfectants.

This study compared the activity of commercial liquid sterilants and disinfectants on Bacillus subtilis spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material. Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization. These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied. Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.

Comparative sensitivity of 13 species of pathogenic bacteria to seven chemical germicides.

BACKGROUND: The relative resistance of diverse human bacterial pathogens to commonly used germicidal agents has not been established. METHODS: We measured by titration the survival of thirteen different bacteria after exposure to glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate, sodium hypochlorite, or phenol. RESULTS: Our comparative experiments allowed classification of the organisms' survival into four groups: (a) Pseudomonas aeruginosa and Staphylococcus aureus showed the most resistance, (b) Clostridium perfringens, Salmonella typhimurium, Staphylococcus epidermidis, and Escherichia coli O157:H7 showed intermediate resistance, (c) Listeria monocytogenes, Shigella sonnei, and Vibrio parahaemolyticus survived some treatments with chemical agents only in the presence of protecting protein (serum albumin), and (d) Vibrio cholerae, Vibrio vulnificus, Bacillus cereus, and Yersinia enterocolitica did not survive any of the treatments applied. CONCLUSION: We found species that more frequently survived exposure to germicidal agents were also those most commonly reported in association with hospital infections. Our findings suggest that resistance to disinfectants may be more important than pathogenicity in determining the relative prominence of an organism as an agent responsible for nosocomial infections.

Mechanism of copper-mediated inactivation of herpes simplex virus.

The inactivation of herpes simplex virus (HSV) by copper was enhanced by the following reducing agents at the indicated relative level: ascorbic acid >> hydrogen peroxide > cysteine. Treatment of HSV-infected cells with combinations of Cu(II) and ascorbate completely inhibited virus plaque formation to below 0.006% of the infectious virus input, while it maintained 30% viability for the host mammalian cells. The logarithm of the surviving fraction of HSV mediated by 1 mg of Cu(II) per liter and 100 mg of reducing agent per liter followed a linear relationship with the reaction time, in which the kinetic rate constant for each reducing agent was -0.87 min(-1) (r = 0.93) for ascorbate, -0.10 min(-1) (r = 0.97) for hydrogen peroxide, and -0.04 min(-1) (r = 0.97) for cysteine. The protective effects of metal chelators and catalase, the lack of effect of superoxide dismutase, and the partial protection conferred by free-radical scavengers suggest that the mechanism of copper-mediated HSV inactivation is similar to that previously reported for copper-mediated DNA damage. The sensitivity exhibited by HSV to Cu(II) and reducing agents, particularly ascorbate, might be useful in the development of therapeutic antiviral agents.

Molecular characteristics of Junin virus.

Results suggest that protein, glycerophospolipid, galactoside, and sialyl glycoside residues are present in Junin virus (JV), are accessible to enzymatic digestion, and play an important role in infection. Four major protein bands with molecular masses (Mr) 64 +/- 2, 56 +/- 2, 52 +/- 3 (mean +/- standard deviation, n = 4) and approximately 12-18 kDa were consistently detected after denaturing gel electrophoresis analysis of purified attenuated JV. The 52 kDa protein showed a diffuse tail in the 52-56 kDa range and was considered to be the JV glycoprotein. By Western blotting, the 64 kDa protein bound a JV neutralizing antibody and was considered to be the viral nucleoprotein. Additional bands corresponding to larger proteins (approximately 200, 96, 86, and 78 80 kDa in size), as well as fainter and broader bands in the 23-44 kDa region were also present in purified JV preparations. The relative resistance of virus infectivity to RNase digestion demonstrates that the genome of JV is protected from enzymatic attack. Analysis of purified JV virions by electrophoresis resolved the viral small (S) RNA and large (L) RNA species, 3636 +/- 54 bases and 7667 +/- 154 bases long, respectively (average length +/- range, in four determinations). The (S) RNA of attenuated JV appeared slightly larger than that reported for a pathogenic strain, ruling out a large sequence deletion as a reason for attenuation.

Effects of salt and serum on the sporicidal activity of liquid disinfectants.

This study compares the effects of various concentrations of salt or serum in the killing of Bacillus subtilis spores by either glutaraldehyde, sodium hypochlorite, cupric ascorbate, hydrogen peroxide, peracetic acid, formaldehyde, or phenol. Salt affected only glutaraldehyde, its sporicidal activity increasing with an increase in concentration of sodium bicarbonate or sodium chloride. The sporicidal activity of glutaraldehyde was minimal when the concentrations of aldehyde groups and lysine residues from protein were similar. We present an equation describing the effect of serum on spore survival as a function of glutaraldehyde concentration that fits the data with a regression coefficient of 0.9. Cupric ascorbate and peracetic acid were inhibited by serum, but this effect was linked to a rise in pH. Sodium hypochlorite was the agent most sensitive to protein, with its sporicidal activity nearly disappearing in the presence of 2% serum or an equivalent amount of purified protein.

Comparative sporicidal effect of liquid chemical germicides on three medical devices contaminated with spores of Bacillus subtilis.

BACKGROUND: The relatively limited variety of surfaces and geometries challenged in current sporicidal testing reduces the predictive value of these analyses when extrapolated to the wide variety of medical devices. The unknown spore load being challenged and the qualitative nature (growth/no growth) of those tests further prevent precise comparison among liquid chemical disinfectants. Hence, the relative activity of different chemical substances has not been clearly established, hindering selection of the best agent for each clinical situation. METHODS: A micromethod was developed to assess sporicidal activity against Bacillus subtilis spores deposited on three different medical devices; carbon steel dental burs, silicone-rubber medical catheters, and titanium-alloy dental abutment screws. The spore load on each device and the recovery after three analytical steps were quantitatively assessed with spores radiolabeled with carbon 14 methionine. RESULTS: The killing of 2 to 7 x 10(6) spores loaded on three different devices and exposed to glutaraldehyde, formaldehyde, copper ascorbate, hydrogen peroxide, peracetic acid, sodium hypochlorite, or phenol for 30 minutes at 20 degrees C ranged from a 10(3)-fold decrease for 10% hydrogen peroxide to zero decrease for 5% phenol. Our results suggest that the nature of the surface being challenged may affect the sporicidal activity of some chemical agents. CONCLUSION: The quantitative data presented allow comparison of the sporicidal effect of different liquid chemical agents. These findings may help prevent an overestimation of sporicidal activity and possible transmission of pathogens from the surface of improperly decontaminated medical devices.

Cupric and ferric ions inactivate HIV.

Human immunodeficiency virus (HIV-1) was inactivated by either cupric or ferric ions when the virus was free in solution and also 3 hr after cell infection. Fifty percent inactivation of cell-free HIV was achieved with Cu(II) at a concentration between 0.16 and 1.6 mM, or by 1.8 to 18 mM Fe(III). Thus, the dose to inactivate 50% of infectious HIV (D50) by Cu(II) or Fe(III) is higher than that reported for glutaraldehyde (0.1 mM); between the D50 reported for sodium hypochlorite (1.3 mM) and sodium hydroxide (11.5 mM), and significantly lower than that required for HIV inactivation by ethanol (360 mM). Treatment of infected cells for 30 min at 20 degrees C with 6 mM Cu(II) or Fe(III) completely inhibited the formation of syncytia and the synthesis of virus-specific p24 antigen in HIV-infected cells, while still preserving cell viability. The virucidal properties of cupric and ferric ions could be exploited for the development of novel virucidal formulations efficient against HIV.

Comparative sporicidal effects of liquid chemical agents.

We compared the effectiveness of glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate (plus a sublethal amount of hydrogen peroxide), sodium hypochlorite, and phenol to inactivate Bacillus subtilis spores under various conditions. Each chemical agent was distinctly affected by pH, storage time after activation, dilution, and temperature. Only three of the preparations (hypochlorite, peracetic acid, and cupric ascorbate) studied here inactivated more than 99.9% of the spore load after a 30-min incubation at 20 degrees C at concentrations generally used to decontaminate medical devices. Under similar conditions, glutaraldehyde inactivated approximately 90%, and hydrogen peroxide, formaldehyde, and phenol produced little killing of spores in suspension. By kinetic analysis at different temperatures, we calculated the rate of spore inactivation (k) and the activation energy of spore killing (delta E) for each chemical agent. Rates of spore inactivation had a similar delta E value of approximately 20 kcal/mol (ca.83.68 kJ/mol) for every substance tested. The variation among k values allowed a quantitative comparison of liquid germicidal agents.

Association between 8-hydroxy-2'-deoxyguanosine formation and DNA strand breaks mediated by copper and iron.

Oxidative DNA damage is involved in diverse biological phenomena and consists of several kinds of lesions, mainly, strand breaks, base modifications, and DNA-protein crosslinking. However, little is known about the existence of a chemical relationship among them or the ratio by which these different types of lesions are produced. In the present study we investigated whether a relationship exists between DNA strand breakage and base modification. We selected cupric [Cu(II)] and ferric [Fe(III)] ions for this study because these transition metals are active catalysts of DNA damage in vivo. Supercoiled plasmid DNA pZ189 was treated with Cu(II) or Fe(III) in the presence of different reducing agents. We measured in each sample both the number of DNA single-strand breaks (SSB) by quantitative electrophoresis and the amount of a modified DNA base, 8-hydroxy-2'-deoxyguanosine (8-OHdG) by HPLC with simultaneous electrochemical (EC) and spectrophotometric detection. The number of DNA SSBs produced was linearly related to the number of 8-OHdG present. The regression of the number of SSBs as a function of the number of 8-OHdG is expressed by the equation [SSBs] = b x [8-OHdG], where b = 1.7, 2.0, 2.7, 1.7, and 9.4, for Cu(II) in the presence of H2O2, L-cysteine and L-ascorbate, and for Fe(III) in the presence of H2O2 and L-ascorbate, respectively. The linear correlation we observed between the production of SSB and 8-OHdG mediated by Fe(III) and by Cu(II) suggests that these products may arise via a common chemical mechanism and could allow an easier and more precise estimation of DNA breakage.

Cytotoxic and mutagenic effects of ferric nitrilotriacetate on L5178Y mouse lymphoma cells.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis that leads to a high incidence of renal adenocarcinoma in rodents. Others have shown that Fe-NTA induces modified DNA base products both in vitro and in vivo. However, Fe-NTA is negative in the Ames Salmonella test with or without S9 activation. The goal of this project was to determine if Fe-NTA is cytotoxic and mutagenic using the L5178Y (TK +/-) mouse lymphoma assay. Our experiments showed a relationship between the concentration of Fe-NTA (0 to 1 mM) and the decrease in relative survival. An exposure-dependent increase in the number of mutations was observed with increasing concentrations of Fe-NTA. At 14% relative survival, there was about a 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells. Ferric nitrate or nitrilotriacetic acid alone induced a relatively low 1.5- or 1.1-fold increase in mutation, respectively. Our results establish that Fe-NTA is mutagenic in the L5178Y mouse lymphoma assay system.

Increased 8-hydroxydeoxyguanosine in kidney and liver of rats continuously exposed to copper.

Copper is a ubiquitous metal in the environment, it is a component of dental casting gold alloys and dental amalgams, and it is a main component in some intrauterine contraceptive devices (IUDs). Since copper materials implanted in the human body corrode and release ions into the surrounding tissue, the potential toxicity caused by contact of this metal with bodily fluids needs to be evaluated. We implanted male Wistar rats with osmotic mini pumps that continuously administered saline, CuCl2, or a copper chelate, cupric nitrilotriacetate (Cu-NTA), at a rate of 4 mg copper/kg body wt/day. This experimental design maintained serum copper concentrations at a level 30-70% (CuCl2) or 100-120% (Cu-NTA) higher than in untreated controls. At different times postimplantation, we measured the levels of 8-hydroxydeoxyguanosine (8-OHdG) in DNA of kidney, liver, and tissue surrounding the pump implant, since production of 8-OHdG has been associated with mutagenesis and carcinogenesis. Hepatic and renal levels of 8-OHdG in CuCl2- or Cu-NTA-treated animals were significantly higher than in control animals. In contrast, histopathologic changes in kidneys and livers of rats exposed to CuCl2 and Cu-NTA were limited to mild changes involving hepatic focal necrosis and slightly increased mitotic activity in the renal proximal tubules. These observations suggest that levels of 8-OHdG could be an early marker of copper toxicity. It is unlikely that the high levels of copper at which we observed DNA modification will be encountered after occupational or environmental exposure. A different situation could be found around medical devices that include copper, particularly IUDs, where the amount of copper administered in our experiments could be released in the uterus of women after a few months of continued IUD use.