Comparative effects of dieldrin on hepatic ploidy, cell proliferation, and apoptosis in rodent liver.

Dieldrin-induced hepatocarcinogenesis, which is seen only in the mouse, apparently occurs through a nongenotoxic mechanism. Previous studies have demonstrated that dieldrin induces hepatic DNA synthesis in mouse, but not rat liver. A number of nongenotoxic hepatocarcinogens have been shown to increase hepatocyte nuclear ploidy following acute and subchronic treatment in rodents, suggesting that an induction of hepatocyte DNA synthesis may occur without a concomitant increase in cell division. The current study examined the effects of dieldrin on changes in hepatocyte DNA synthesis, mitosis, apoptosis, and ploidy in mouse liver (the sensitive strain and target tissue for dieldrin-induced carcinogenicity) and the rat liver (an insensitive species). Male F344 rats and B6C3F1 mice were treated with 0, 1, 3, or 10 mg dieldrin/kg diet and were sampled after 7, 14, 28, or 90 d on diet. Liver from mice fed 10 mg dieldrin/kg diet exhibited significantly increased DNA synthesis and mitosis at 14, 28, or 90 d on diet. In rats, no increase in DNA synthesis or mitotic index was observed. The apoptotic index in liver of mice and rats did not change over the 90-d study period. Exposure of mice to only the highest dose of dieldrin produced a significant increase in octaploid (8N) hepatocytes and a decrease in diploid (2N) hepatocytes, which were restricted primarily to centrilobular hepatocytes, with the periportal region showing little or no change from control. No changes in hepatocyte nuclear ploidy were observed in the rat. This study demonstrates that exposure to high concentrations of dieldrin is accompanied by increased nuclear ploidy and mitosis in mouse, but not rat, liver. It is proposed that the observed increase in nuclear ploidy in the mouse may reflect an adaptive response to dieldrin exposure.

Monograph: reassessment of human cancer risk of aldrin/dieldrin.

In 1987, the US Environmental Protection Agency (EPA) classified aldrin and dieldrin as category B2 carcinogens, i.e. probable human carcinogens, based largely on the increase in liver tumors in mice fed either organochlorine insecticide. At that date, the relevant epidemiology was deemed inadequate to influence the cancer risk assessment. More time has now elapsed since early exposures of manufacturing workers to aldrin/dieldrin; therefore, updated epidemiological data possess more power to detect exposure-related differences in cancer risk and mortality. Also, recent experimental studies provide a plausible mode of action to explain the mouse specificity of dieldrin-induced hepatocarcinogenesis and call into question the relevance of this activity to human cancer risk. This monograph places this new information within the historic and current perspectives of human cancer risk assessment, including EPA's 1996 Proposed Guidelines for Carcinogen Risk Assessment. Updated epidemiological studies of manufacturing workers in which lifetime exposures to aldrin/dieldrin have been quantified do not indicate increased mortality or cancer risk. In fact, at the middle range of exposures, there is evidence of a decrease in both mortality from all causes and cancer. Recent experimental studies indicate that dieldrin-induced hepatocarcinogenesis in mice occurs through a nongenotoxic mode of action, in which the slow oxidative metabolism of dieldrin is accompanied by an increased production of reactive oxygen species, depletion of hepatic antioxidant defenses (particularly alpha-tocopherol), and peroxidation of liver lipids. Dieldrin-induced oxidative stress or its sequelae apparently result in modulation of gene expression that favors expansion of initiated mouse, but not rat, liver cells; thus, dieldrin acts as a nongenotoxic promoter/accelerator of background liver tumorigenesis in the mouse. Within the framework of EPA's Proposed Guidelines for Carcinogen Risk Assessment, it is proposed that the most appropriate cancer risk descriptor for aldrin/dieldrin, relating to the mouse liver tumor response, is 'not likely a human carcinogen', a descriptor consistent with the example of phenobarbital cited by EPA.

Lack of effect of a 60 Hz magnetic field on biomarkers of tumor promotion in the skin of SENCAR mice.

It has been proposed that extremely low frequency magnetic fields may enhance tumorigenesis through a co-promotional mechanism. This hypothesis has been further tested using the two-stage model of mouse skin carcinogenesis, i.e. 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of skin carcinogenesis in mice initiated by a single subcarcinogenic dose of 7,12-dimethylbenz[a]anthracene. Experimentation utilized three different doses of TPA within its dose-response range (0.85, 1.70 or 3.40 nmol) and examined the following early biomarkers of tumor promotion after 1, 2 and 5 weeks of promotion: increases in epidermal thickness and the labeling index of epidermal cells, induction of epidermal ornithine decarboxylase activity and down-regulation of epidermal protein kinase C activity. Mice exposed to a 60 Hz magnetic field having a flux density of 2 mT for 6 h/day for 5 days/week were compared with mice exposed to an ambient magnetic field. Within the sensitivity limits of the biomarker methodology and the exposure parameters employed, no consistent, statistically significant effects indicative of promotion or co-promotion by the magnetic field were demonstrated.

Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates.

Topical application of certain petroleum middle distillates (PMD) to mice produces skin tumors after long latency, and initiation/promotion protocols indicate that this effect is associated with their tumor promoting activity. Since induction of sustained, potentiated epidermal hyperplasia is predictive of promoting activity, five compositionally distinct PMD [hydrodesulfurized kerosene (API 81-07); hydrodesulfurized PMD (API 81-10); odorless light petroleum hydrocarbons; severely hydrotreated light vacuum distillate (LVD); and lightly refined paraffinic oil (LRPO)] were assessed for their effects on epidermal hyperplasia. PMD were administered (2 x/week for 2 weeks) to skin of CD-1 mice. Four quantitative biomarkers of epidermal hyperplasia were evaluated: epidermal thickness, number of nucleated epidermal cells per unit length of basement membrane, labeling (BrdUrd) index of epidermal cells, and induction of epidermal ornithine decarboxylase (ODC) activity. As positive controls, 12-O-tetradecanoylphorbol-13-acetate (TPA) and n-dodecane were utilized. PMD-induced skin irritation was evaluated visually and/or histopathologically. All five PMD produced dose-dependent, skin irritation and epidermal hyperplasia. On a weight basis the magnitude of the maximal PMD-induced effects was similar to that produced by n-dodecane, but > 1000-fold less than that produced by TPA. Epidermal hyperplasia and subacute skin irritancy produced by the five PMD were similar. Of the four short-term markers of tumor promotion assessed, labeling index and epidermal ODC activity were predictive of the relative promoting activities of those PMD for which tumorigenicity bioassay data are available, i.e., API 81-07 > API 81-10 > LRPO. An apparent discrepancy to the predictability of epidermal ODC activity occurred with LRPO:toluene [1:1 (v/v)]. This mixture is nontumorigenic, yet significantly induced epidermal ODC activity. This mixture, however, produced severe epidermal toxicity that precluded any meaningful analysis of short-term biomarkers in relationship to biological activity.

Lack of a co-promoting effect of a 60 Hz magnetic field on skin tumorigenesis in SENCAR mice.

It has been proposed that extremely low frequency (ELF) magnetic fields may enhance tumorigenesis through a co-promotional mechanism. This hypothesis has been further tested using the two-stage model of mouse skin carcinogenesis, i.e. 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of skin tumors in mice initiated by a single subcarcinogenic dose of 7,12-dimethylbenz[a]anthracene. Experimentation described herein utilized the SENCAR mouse and examined the effect of a magnetic field on skin tumor promotion induced by three different doses of TPA within its dose-response range, i.e. 0.85, 1.70 or 3.40 nmol, administered twice per week. SENCAR mice (56/treatment group) were exposed to a 60 Hz magnetic field having a flux density of 2 mT for 6 h/day for 5 days/week and compared with mice exposed to the ambient magnetic field. Tumor incidence and multiplicity were monitored weekly for 23 weeks of TPA promotion. Statistical evaluation of the effects of the magnetic field on tumor incidence and multiplicity did not reveal any statistically significant effects; thus, within the sensitivity limits imposed by the animal model and the exposure parameters employed, no promotional or co-promotional effect of a 2 mT magnetic field on skin tumor development in SENCAR mice could be demonstrated.

Role of oxidative stress in the selective toxicity of dieldrin in the mouse liver.

Dieldrin, an organochlorine insecticide, induces hepatic tumors in mice but not in rats. Although the mechanism(s) responsible for this species specificity is not fully understood, accumulating evidence indicates that oxidative stress may be involved. This study examined the association of dieldrin-induced hepatic DNA synthesis with the modulation of biomarkers of oxidative damage to lipids (malondialdehyde [MDA]) and DNA (8-hydroxy-2-deoxyguanosine [oh8dG]), in male B6C3F1 mice and F344 rats fed dieldrin (0.1, 1.0, or 10 mg/kg diet) for 7, 14, 28, and 90 days. The nonenzymatic components of the antioxidant defense system (ascorbic acid, glutathione, and alpha-tocopherol) were also examined. Increased urinary MDA was observed in mice fed 0.1, 1.0, or 10 mg dieldrin/kg diet for 7, 14, 28, and 90 days; while increased hepatic MDA was seen only after 7 days in mice fed 0.1, 1.0, or 10 mg dieldrin/kg diet and after 14 days in mice fed 10 mg/kg diet. In rats, dieldrin had no effect on either hepatic MDA or urine MDA levels after 7, 14, and 28 days of treatment. A dose-dependent increase in urinary MDA was observed in rats at the 90-day sampling time. The only significant elevation in urinary or hepatic oh8dG content was limited to urinary oh8dG in mice fed 10 mg/kg dieldrin diet for 14 days. Dietary dieldrin produced sustained decreases in hepatic and serum alpha-tocopherol and sustained elevations in hepatic ascorbic acid in both mice and rats. Rats, however, possessed a three- to four-fold higher content of endogenous or basal (control) hepatic alpha-tocopherol; and, even when fed 10 mg dieldrin/kg diet, the levels of hepatic alpha-tocopherol were maintained at higher levels than those of mice fed control diet. In both rats and mice fed dieldrin, transient (14 and 28 days on diet) elevations in hepatic glutathione were observed. These data support the hypothesis that the species specificity of dieldrin-induced hepatotoxicity may be related to dieldrin's ability to induce oxidative stress in the liver of mice, but not in rats. Only in mice fed dieldrin was a temporal association of increases in hepatic MDA content and hepatic DNA synthesis seen, suggesting that oxidative damage (shown by increased lipid peroxidation) may be involved in early events in dieldrin-induced hepatocarcinogenesis. Rats may be protected from dieldrin-induced oxidative stress by a more effective antioxidant defense system, characterized by higher basal levels of hepatic alpha-tocopherol and ascorbic acid than that seen in the mouse.

Vitamin E modulation of dieldrin-induced hepatic focal lesion growth in mice.

The effect of vitamin E on dieldrin-induced hepatic focal lesion growth in male B6C3F1 mice previously treated with diethylnitrosamine (DEN) was investigated. After hepatic focal lesions were formed, mice were placed into one of the following treatment groups: Group 1, 50 mg vitamin E/kg diet (control NIH-07 diet); Group 2, 10 mg dieldrin/kg NIH-07 diet; Group 3, 10 mg dieldrin and 450 mg vitamin E/kg NIH-07 diet; and Group 4, 450 mg vitamin E/kg NIH-07 diet. Mice were killed and necropsied after 30 and 60 d of dietary treatment. The effect of treatment on lesion growth was examined by measuring the number of focal lesions per liver and the relative hepatic focal lesion volume. In addition, the possible cellular mechanism of focal hepatocyte growth was investigated by examining both focal DNA synthesis and apoptosis. Dieldrin treatment alone (Group 2) increased the focal lesion volume, focal lesion number, and focal lesion labeling index. Supplementation with vitamin E (Group 3) blocked this effect. Vitamin E supplementation to the diet alone (Group 4) also enhanced focal lesion growth and increased the number of lesions per liver, the relative focal volume, and the labeling index in hepatic focal lesions. Interestingly, vitamin E supplementation inhibited apoptosis in normal liver but did not produce an observable decrease in apoptosis in hepatic focal lesions. The present study showed that dieldrin (Group 2) or vitamin E supplementation alone (Group 4) promoted the growth of hepatic focal lesions in mice. However, when vitamin E is supplemented to dieldrin-fed mice (Group 3), there is an inhibition of hepatic focal lesion growth.

The role of oxidative stress in chemical carcinogenesis.

Oxidative stress results when the balance between the production of reactive oxygen species (ROS) overrides the antioxidant capability of the target cell; oxidative damage from the interaction of reactive oxygen with critical cellular macromolecules may occur. ROS may interact with and modify cellular protein, lipid, and DNA, which results in altered target cell function. The accumulation of oxidative damage has been implicated in both acute and chronic cell injury including possible participation in the formation of cancer. Acute oxidative injury may produce selective cell death and a compensatory increase in cell proliferation. This stimulus may result in the formation of newly initiated preneoplastic cells and/or enhance the selective clonal expansion of latent initiated preneoplastic cells. Similarly, sublethal acute oxidative injury may produce unrepaired DNA damage and result in the formation of new mutations and, potentially, new initiated cells. In contrast, sustained chronic oxidative injury may lead to a nonlethal modification of normal cellular growth control mechanisms. Cellular oxidative stress can modify intercellular communication, protein kinase activity, membrane structure and function, and gene expression, and result in modulation of cell growth. We examined the role of oxidative stress as a possible mechanism by which nongenotoxic carcinogens may function. In studies with the selective mouse liver carcinogen dieldrin, a species-specific and dose-dependent decrease in liver antioxidant concentrations with a concomitant increase in ROS formation and oxidative damage was seen. This increase in oxidative stress correlated with an increase in hepatocyte DNA synthesis. Antioxidant supplementation prevented the dieldrin-induced cellular changes. Our findings suggest that the effect of nongenotoxic carcinogens (if they function through oxidative mechanisms) may be amplified in rodents but not in primates because of rodents' greater sensitivity to ROS. These results and findings reported by others support a potential role for oxidative-induced injury in the cancer process specifically during the promotion stage.

Role of oxidative stress in the mechanism of dieldrin's hepatotoxicity.

The production of reactive oxygen species (ROS) by toxic chemicals has been implicated in acute and chronic disease states, including cancer. This increase in cellular ROS can lead to a state of oxidative stress. Many compounds selectively induce hepatic tumors in mice but not rats. The mechanism for the induction of hepatic cancer by these compounds and the observed species selectivity of this effect are not known but may be related to the induction of oxidative stress. Dieldrin is one such compound and is used in the present study to characterize the relationship between oxidative stress and the observed selective hepatotoxicity of dieldrin in mice. It was found that dieldrin induced oxidative stress in the mouse but not the rat, and the observed oxidative stress correlated with the induction of DNA S-phase synthesis. This evidence suggests that the induction of oxidative stress may be a mechanism by which dieldrin and other mouse specific compounds selectively induce their hepatic toxic effects in mice.

Reversibility of promoter induced hepatic focal lesion growth in mice.

The effect of cessation of phenobarbital and dieldren treatment on hepatic focal lesion growth in male B6C3F1 mice was investigated. Following induction of lesions by diethylnitrosamine, mice were placed on control NIH-07 diet (control diet) or NIH-07 diet containing either dieldrin (10.0 mg/kg diet) or phenobarbital (500 mg/kg diet). Mice were sacrificed after 30 and 60 days of dietary treatment. Two additional groups of mice were fed either the dieldren- or phenobarbital-containing diet for 30 days followed by feeding of NIH-07-only diet for an additional 30 days. The effect of treatment and removal of dieldrin or phenobarbital on lesion growth was examined by measuring both the number of focal lesions per liver and the relative volume of focal lesions. In addition, the rate of cell proliferation and programmed cell death in focal lesion growth was investigated by examining DNA synthesis and apoptosis in the focal lesions. Dietary dieldrin or phenobarbital increased the number of focal lesions and the focal lesion volume. In both dieldrin- and phenobarbital-treated mice, an increased number of eosinophilic lesions were seen. The focal lesion volume was increased in both eosinophilic and basophilic lesions. Dieldrin and phenobarbital treatment also increased the DNA synthetic labeling index in both eosinophilic and basophilic lesions. Removal of dieldrin or phenobarbital from the diet after 30 days of promoter treatment decreased the total number and volume of hepatic focal lesions. The labeling index of the focal lesions was also decreased in these mice. At the terminal sacrifice, the percentage of apoptotic cells in focal lesions was higher in mice fed dieldrin- or phenobarbital-containing diets for the entire 60 days than in mice returned to control diet for the last 30 days. Eosinophilic lesions were more dependent on the presence of a promoting stimulus than the basophilic lesions. These data indicate that induction and maintenance of the growth of some preneoplastic lesions in the mouse may be dependent upon continuous tumor promoter treatment.

Selective dieldrin promotion of hepatic focal lesions in mice.

Chronic exposure to a number of chlorinated pesticides, including dieldrin, results in an increased incidence and/or multiplicity of hepatocellular neoplasia in mice, with no such effect in similarly treated rats. One possible explanation of this observed selective carcinogenicity is species-specific hepatic tumor promotion. In the present study we examined the dose-response effect of dieldrin (at several doses) on focal lesion growth (tumor promotion), hepatocyte apoptosis and DNA synthesis in rat and mouse liver. Preneoplastic focal hepatic lesions were produced by diethylnitrosamine (DEN). After the lesions developed, mice and rats were placed into one of the following dose groups: control (NIH-07 diet) or 0.1, 1.0 or 10.0 mg dieldrin/kg diet. Increased focal lesion volume, number of foci per liver and focal DNA synthetic labeling index were observed in 10 mg dieldrin/kg diet-treated mice, but not in similarly treated rats. Dieldrin at dietary concentrations of 0.1 and 1.0 mg/kg diet produced an increase in the number of preneoplastic lesions (0.1 mg/kg diet at 7 days only) and focal volume (0.1 mg/kg diet at 7 and 30 days, 1.0 mg/kg diet at 30 days), but these concentrations did not increase focal DNA labeling index. At dietary concentrations of 0.1, 1.0 and 10 mg dieldrin/kg diet no significant change in lesion percent volume, number of preneoplastic lesions per liver or preneoplastic lesion DNA labeling index was seen in treated rats compared with control rats. Apoptosis, a form of programed cell death, was not decreased in foci by any concentration of dieldrin in either rats or mice. Thus our results suggest that dieldrin may function as a mouse-specific tumor promoter through increased lesion DNA synthesis.

Dose dependence of phenobarbital promotion of preneoplastic hepatic lesions in F344 rats and B6C3F1 mice: effects on DNA synthesis and apoptosis.

Phenobarbital (PB), a non-genotoxic hepatocarcinogen in rodents, has been studied extensively but its mechanism of carcinogenic action is unclear. PB appears to function as a tumor promoter by selectively inducing the growth of preneoplastic hepatocytes. In the present study, the comparative effects of PB at tumor-promoting and non-promoting doses were examined in male B6C3F1 mice and male F344 rats. In addition, the mechanism by which PB produced the selective induction of preneoplastic cell growth (increased DNA synthesis/cell proliferation and/or decreased apoptosis) was investigated. Preneoplastic focal lesions were produced using diethylnitrosamine (DEN). After the lesions were histologically apparent, mice and rats were fed PB (10, 100, or 500 mg/kg NIH-07 diet) or control diet and sampled after 7, 30 and 60 days of treatment In both mice and rats, 100 and 500 mg PB/kg increased the number and the relative volume of focal lesions. In rats and mice, 10 mg PB/kg did not enhance focal lesion growth. The preneoplastic lesions that clonally expanded due to phenobarbital treatment were predominantly eosinophilic in appearance. In addition, DNA synthesis in focal hepatocytes was significantly increased in the 100 and 500 mg PB/kg diet. In PB-treated mice and rats, there also was a significant decrease in the rates of apoptosis in focal hepatocytes. Therefore, our data showed that PB at doses of 100 and 500 mg/kg diet promoted focal hepatic lesion growth both by increasing DNA synthesis and cell proliferation and by decreasing the rate of apoptosis.

Subchronic effects of dieldrin and phenobarbital on hepatic DNA synthesis in mice and rats.

Dieldrin, an organochlorine pesticide, has been shown to be hepatocarcinogenic in mice but not rats. Phenobarbital, in contrast, induces hepatic tumors in both mice and rats. Previous studies have shown that acute dietary exposure of rats or mice to either dieldrin or phenobarbital produces several liver changes, including centrilobular hypertrophy, induction of hepatic cytochrome P450, and increased liver weight. The present study examined the subchronic effect of dieldrin (0.1, 1.0, 3.0, 10.0 mg dieldrin/kg diet) and phenobarbital (10, 50, 100, 500 mg phenobarbital/kg diet) on the induction of hepatic DNA synthesis and hepatocyte lethality in male B6C3F1 mice and male F344 rats. Eight-week-old animals were treated as above and evaluated for hepatic DNA synthesis after 7, 14, 21, 28, and 90 days of continual treatment to dieldrin or phenobarbital. Maximal induction of hepatic DNA synthesis in mice was seen at the 14-, 21-, and 28-day sampling times. In rats, no significant increase in hepatic DNA synthesis or hepatocyte lethality was observed at any dose of dieldrin investigated. Phenobarbital produced a significant increase in hepatic DNA synthesis in both rat and mouse liver following 7 days of treatment. The induction of DNA synthesis in rat liver was transient, with the labeling index returning to control levels by 14 days of treatment. In contrast, mice treated with phenobarbital showed a significant increase in hepatic DNA synthesis throughout the treatment. In both mice and rats, dieldrin and phenobarbital induced hepatic DNA synthesis selectively in the centrilobular region of the hepatic lobule. The lack of an increase in serum enzymes indicative of hepatic damage and the absence of liver histopathology in mice or rats fed dieldrin or phenobarbital indicate that the induction of DNA synthesis was not mediated by a cytolethal, compensatory hyperplastic response, suggesting a mitogenic mechanism. Therefore, the species-specific induction of hepatic DNA synthesis by either dieldrin or phenobarbital correlated with the previously observed species-specific induction of hepatic cancer by these two compounds.

Oxidative stress in nongenotoxic carcinogenesis.

The induction of oxidative stress in the target tissue has been proposed as a possible mechanism of action for nongenotoxic carcinogens. A variety of nongenotoxic hepatocarcinogens including peroxisome proliferators, organochlorines, barbiturates, and metals have been shown to produce an increase in reactive oxygen species (ROS) in the liver. Our group has examined the induction of oxidative stress by the organochlorine mouse hepatic carcinogen, dieldrin. Using a salicylate spin trap assay, dieldrin was found to produce mouse liver-specific increases in ROS in cultured hepatocytes. Increased amounts of hepatic 8-hydroxy-2'-deoxyguanosine and malondialdehyde (MDA) and decreased levels of cellular antioxidants were also seen in cultured mouse hepatocytes following dieldrin treatment. In subchronically dieldrin-treated mice and rats, hepatic vitamin E (Vit E) was decreased correlated with dieldrin dose. While Vit E levels were decreased in both rats and mice, the normal lower levels of Vit E in the mouse resulted in a subsequent oxidative stress, evidenced by an increase in MDA formation in the mouse liver. Dieldrin also produced a dose-dependent increase in DNA synthesis in the mouse (not the rat) following subchronic treatment. These effects seen in both cells in culture and in vivo were species specific, organ specific, and dose dependent which directly correlated with the observed pattern of cancer induction for dieldrin in rodents (mouse liver-specific). These findings support a possible role for the induction of oxidative stress in nongenotoxic hepatic carcinogenesis possibly through modulation of gene expression.

Induction of short-term biomarkers of tumor promotion in skin of CD-1 mice by petroleum middle distillates: preliminary observations.

The induction of sustained epidermal hyperplasia in mouse skin has been shown to be a reliable predictor of tumor promoting activity for chemicals as diverse as phorbol esters, anthralins, n-dodecane and organic peroxides (DiGiovanni, 1992). The results contained herein demonstrate that API 81-07 and API 81-10, petroleum middle distillates that exhibit tumor promoting activity in mouse skin, induce epidermal hyperplasia and ODC activity. Other petroleum middle distillates (odorless light petroleum hydrocarbons, a light vacuum distillate, and a mineral seal oil) were also shown to share these activities. It should be emphasized that the relevance of these observations to human skin remains unresolved; however, the availability of these short-term biomarkers offers the opportunity to address the issue by performing comparative investigations on the effects of petroleum middle distillates on human skin xenografted to athymic (nude) mice. Such studies are being initiated.