The major goal of this research was to test the hypothesis that PHAH exposures increase ROS generation, which in turn results in higher numbers of M1dG adducts in genomic DNA. As an initial approach to test the hypothesis, the effect of TCDD on the number of M1dG adducts was examined using mice treated with single doses of different amounts of TCDD. In a parallel experiment, different combinations of PHAH mixtures were given to mice to evaluate the TEF methodology in their toxicities, including oxidative DNA damage. Mix A consisted of DLC including TCDD. Mix B consisted of non-dioxin like PCB that are major PCB congeners found in the environment. To emulate environmental exposures which include a mixture of PHAH, mix C consisted of a combination of mix A and mix B. The toxicity and the change in the number of M1dG adducts were examined a week after PHAH treatment.
A previous study [28
] had shown that TCDD treatment resulted in a dose-dependent increase in the concentration of malondialdehyde (i.e., a major precursor to M1
dG adduct formation) in the livers of these mice. Cytochrome C reduction (i.e., an indicator of superoxide anion radical production) showed statistically significant increases in mice that received 10 or 100 μg/kg/d of TCDD [28
]. However, as described in , there was no trend in the numbers of M1
dG DNA adducts in response to the different doses of TCDD or one of the PHAH mixtures (non-parametric Jonckheere-Terpstra exact test, p
> 0.18). This result is consistent with the findings from other investigators who reported no significant changes in the numbers of 8-OH-dG adducts after a single exposure to DLC [36
]. In contrast, subchronic or chronic exposures to the chemicals resulted in an accumulation of 8-OH-dG adducts in animals [36
Effect of single dose of TCDD or PHAH mixture on the background level of hepatic M1dG adducts
It has been hypothesized by a number of investigators [12
] that DNA repair in healthy control animals is sufficient to repair DNA adducts formed by an acute exposure to PHAH. Unlike acute exposures, however, long-term exposures to PHAH may change the capacity of DNA repair by modifying the expression of proteins involved in DNA repair, analogous to how PHAH changes the expression of metabolic enzymes [38
]. Additionally, it has been proposed that DNA repair proteins can be impaired by mutations caused by the slow accumulation of DNA adducts over a long period of time [37
]. This argument is supported by the findings from previous studies that reported sustained DNA adduct (i.e., 8-OH-dG) accumulation, toxicity, and carcinogenesis [12
To determine the role of oxidative DNA damage in the toxicity and carcinogenicity of PCBs, the accumulation of M1dG adducts in livers and brain was examined in rats that had been exposed for a year to PCB 153, PCB 126, or a mixture of PCB 153 and PCB 126. As shown in , PCB exposure produced statistically significant changes in the accumulation of hepatic M1dG adducts in the rats exposed to PCB 126 (F(4,35)=13.31, p < .0001) or to the mixture of PCB 126 and PCB 153 in a 1:1000 ratio (F(4,35)=19.44, p < .0001). There was no significant effect of PCB 153 exposure on M1dG DNA adduct accumulation in rat liver (F(4,33)=1.32, p =.2820). Linear regression confirmed dose-dependent increases in the numbers of M1dG adducts in the rats exposed to PCB 126 (R2 = .5851, p < .0001) or to the mixture of PCB 126 and PCB 153 (R2 = .6880, p < .0001). Interestingly, analyses of the linear regression model revealed that the mixture of PCB 126 and PCB 153 had a stronger effect on the accumulation of hepatic M1dG adducts than PCB 126 alone (p = .0013). The response to the mixture of PCB 126 and PCB 153 in the accumulation of hepatic M1dG adducts was even stronger than the sum of the individual response to PCB 126 and PCB 153 (p = .0027). Compared to the control, PCB 126 resulted in significantly increased accumulation of M1dG adducts in rat liver at 1000 ng/kg. However, by co-administrating a 1000-fold excess of PCB 153, PCB 126 produced significantly higher numbers of M1dG adduct in rat liver at 300 ng/kg/ as well as at 1000 ng/kg/d when the numbers were compared to that of control rats. (p = .0274, and p < .0001, respectively). These results provide strong evidence in support of the hypothesis that long-term exposure to PCB causes the accumulation of oxidative DNA lesions that may play a role in toxicity, mutation and cancer development. Furthermore, the results from this study support the hypothesis that PCB 153 which is a non-dioxin like PCB potentiates the PCB 126 mediated DNA damage.
Accumulation of M1dG DNA adducts in rat liver tissues after chronic exposure to PCBs
This hypothesis was further tested in the experiments with rats that were dosed with fixed amounts of PCB 126 (300 ng/kg/d) in combination of different amounts of PCB 153. The effect of PCB treatments on hepatic M1dG adducts is shown in . The number of M1dG adducts in livers for the mixture of PCB 126/PCB 153 at 300/3000 was significantly higher compared to both 300/0 (p < .0001) and 0/3000 (p < .0001) (. A positive linear association (R2 = .603, p < .0001) was confirmed between the number of M1dG adducts in liver and the amount of PCB 153 when the rats were co-administered 300 ng/kg/d PCB 126 (). Overall, these results support the hypothesis that non-dioxin like PHAH can significantly increase DNA damage resulting from simultaneous exposures to DLC.
Accumulation of M1dG DNA adducts in rat liver tissues after chronic exposure to mixtures of PCB 126 and PCB 153
The effect of PCB exposure on the accumulation of M1dG adducts in the brain was examined and the results are described in . There was no trend in the number of M1dG adducts in response to the different doses of either PCB 153 alone or the mixture of PCB 153 and PCB 126 (non-parametric Jonckheere-Terpstra exact test, p > .28). The effect of PCB 126 exposure on M1dG accumulation was not examined because tissue samples were not available.
Analyses of M1dG DNA adducts in brain tissues after chronic exposure to PCBs
Finally, the results from this study were compared to those from the NTP cancer bioassays [30
]. The NTP reported that increasing the proportion of PCB 153 in the mixtures of PCB 126 resulted in increases in cell proliferation in rat liver. Furthermore, there was a positive effect of PCB 153 in the PCB 153/ PCB 126 mixture on liver toxicity, including hepatocyte hypertrophy, cholangiofibrosis, eosinophilic foci, clear cell foci, basophilic foci, diffuse and focal fatty change, bile duct hyperplasia, and hematopoietic cell proliferation. Carcinogenesis was also enhanced after a two-year exposure. A positive effect of increasing the amount of PCB 153 in the mixture was evident in increased incidences of hepatocellular adenoma and cholangiocarcinoma. As described in , the dose response for M1
dG adduct accumulation after PCB exposure is very similar to that of developing neoplastic lesions. The number of M1
dG adducts in liver showed positive correlations with the incidence of hepatocellular adenoma and cholangiocarcinoma. It is noteworthy that the change in the number of hepatic M1
dG adducts was detected after a one-year exposure to PCBs while the increase in cancer incidence was observed after a two-year exposure to PCBs. This result supports the hypothesis that oxidative DNA damage plays an important role in cancer development associated with chronic exposure to PHAHs. Compared to other biomarkers such as cell proliferation and cytochrome P450 expression that have been used in the TEF evaluation for PHAHs, the numbers of hepatic M1
dG adducts showed strong positive correlations to the incidence of neoplastic lesions in the rats treated with the mixture of PCBs.
Dose response for the formation of neoplastic lesions and for M1dG adduct accumulation in female rats treated with mixtures consisting of PCB 126 and PCB 153
PCB153, which does not have a TEF value since it has no dioxin like effects, enhances the accumulation of oxidative DNA lesions in genomic DNA, thus increasing chances for toxicity, mutation and cancer development. Similar to these results, the synergistic effect of PCB 153 with DLC other than PCB 126 on the toxicity of DLC has been previously reported. Van Birgelen [42
] demonstrated a synergistic effect of co-administration of PCB 153 and TCDD on hepatic porphyrin accumulation in female Sprague-Dawley rats fed diets containing these chemicals for 13 weeks. To explore this synergistic effect of PCB 153 on the toxicity of DLC, a number of researchers investigated the role of PCB 153 and found that it increased the hepatic concentrations of DLC [43
]. PCB 153 was shown to enhance the retention of DLC in the cell by inducing both AhR and CYP1A2, the two major binding proteins for DLC [45
]. However, analysis of hepatic PCB 126 concentrations by the NTP as a part of the cancer bioassay, showed that the proportion of PCB 153 in the mixture relative to PCB 126 had no significant effect on the amounts of PCB 126 in liver and lung tissues [30
]. Consistent with this result, hepatic CYP1A1 and CYP1A2 enzyme activities were not affected by the proportion of PCB 153 in mixtures with constant amounts of PCB 126. Therefore, the molecular mechanism involved in this synergistic effect between PCB 126 and PCB 153 on M1
dG adduct accumulation in the liver requires additional research.
In summary, the present study supports the hypothesis that oxidative DNA damage plays an important role in the toxicity and carcinogenicity associated with chronic exposure to PHAHs. The accumulation of M1dG adducts in the tissues were dose- and time-dependent on PHAH exposure, which is similar to the incidence of pre-neoplastic lesions and subsequent cancer development. Enhanced DLC toxicities (i.e., the accumulation of M1dG adducts and pre-neoplastic lesions in liver) by non-dioxin like PHAH suggest that the combination of different types of PHAH can result in greater toxicity than predicted by TEFs. Overall, the findings from the present study provide additional information that may be important in the scientific assessment of risk for complex PHAH mixtures present in the environment.