A comparison of 24 chemicals in the six-well bacterial reverse mutation assay to the standard 100-mm Petri plate bacterial reverse mutation assay in two laboratories.

The bacterial reverse mutation assay (Ames) is a fundamental genetic toxicology test, and efforts to miniaturize the regulatory GLP version are essential in assessing genotoxic liabilities earlier in the drug development pipeline. Two versions of the Ames were compared: the six-well (miniaturized) plate and the standard 100-mm plate test at two different laboratories. Of twenty-four chemicals tested, a subset of six chemicals was tested in the six-well test only and the remaining eighteen were evaluated in both versions of the test. The plate incorporation procedure was used with one Escherichia coli and four different Salmonella strains. The six-well test uses the same plating procedure and evaluation methods as the standard Ames assay in 100-mm plates, but the smaller format requires 20% of the test chemical. Additionally, the six-well test uses a limit concentration of 1000 µg/well versus the standard Petri plate test limit concentration of 5000 µg/plate. Testing across the two formats resulted in 100% concordance in overall mutagenicity judgement and 94% concordance across all tester strains and conditions. Known mutagenic positive control chemicals were correctly detected as positive in both formats. The overall conclusion is that the six-well assay results are concordant with the standard assay format in this evaluation and could be a reliable alternative.

Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations.

Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC50 in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 µM concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 µM. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity.

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.

Dose-response examination of UDP-glucuronosyltransferase inducers and their ability to increase both TGF-beta expression and thyroid follicular cell apoptosis.

Exposure to certain microsomal enzyme inducers that increase UDP-glucuronosyltransferase (UDP-GT) activity decreases thyroid hormone levels, which may lead to a subsequent increase in thyroid-stimulating hormone (TSH). This elevation of serum TSH has many effects on the thyroid, including increasing thyroid follicular cell proliferation, leading to hyperplasia. While induction of UDP-GT activity decreases thyroid hormone levels by enhancing biotransformation and subsequent biliary secretion, only certain UDP-GT inducers exhibit the ability to increase serum TSH levels. For example, phenobarbital (PB) and pregnenolone-16alpha-carbonitrile (PCN) increase serum levels of TSH, while 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB) do not. Increased serum TSH concentration also enhances thyroid gland expression of TGF-beta1, an anti-proliferative, pro-apoptotic protein. In a previous study in our laboratory, rats were treated for various times (up to 90 days) with PB and PCN, which increased TGF-beta1 protein and apoptosis. The present study was designed to examine the dose-response effect of TSH-increasing (PB and PCN) and nonincreasing (3MC and PCB) UDP-GT inducers on apoptosis and TGF-beta1. PB and PCN, UDP-GT inducing compounds which increase serum TSH, increased the percentage of TGF-beta1-positive follicular cells and increased apoptosis. In contrast, UDP-GT inducers that did not increase TSH (3MC and PCB) did not alter cell death or TGF-beta production. These data suggest that the increase of TGF-beta by TSH may serve to regulate the growth of hyperplastic thyroid.

Inhibition of gap-junctional-intercellular communication in thyroid-follicular cells by propylthiouracil and low iodine diet.

Propylthiouracil (PTU) or low-iodine diet (LID) treatment increases thyroid-follicular-cell proliferation, possibly by disrupting the movement of small molecules (< 1.2 kD) through membrane channels called gap junctions. Numerous tumor promoters and proliferative disease states exhibit inhibited gap-junctional-intercellular communication (GJIC) prior to the induction of cell proliferation, yet the association between GJIC and apoptosis is unclear. In the present study, we used an ex vivo method to examine whether GJIC is inhibited in the thyroid of PTU- or LID-treated rats. In addition, the effect of these models of hypothyroidism on thyroid-follicular-cell proliferation and apoptosis was examined to determine the association between GJIC and cell homeostasis. After 14 days of treatment of either PTU or LID (plus 1% KClO4 in the drinking water), serum tri-iodothyronine (T3) and thyroxine, (T4) was decreased to nearly undetectable levels and serum TSH was increased in PTU- and LID-treated rats. At the same time point, GJIC was decreased 30-35% in PTU- and LID-treated rats while thyroid-follicular-cell proliferation increased nearly threefold in both treatment groups. Interestingly, apoptosis increased twofold in both hypothyroid treatment groups. These data suggest that PTU or LID treatment inhibit thyroid GJIC during a state of increased thyroid-follicular-cell proliferation and apoptosis. While the increase in proliferation was anticipated, the paradoxical increase in apoptosis during decreased GJIC in thyroid-follicular cells warrants further examination.

Inhibition of gap-junctional-intercellular communication in intact rat liver by nongenotoxic hepatocarcinogens.

Many nongenotoxic hepatocarcinogens can induce cell proliferation, and inhibit apoptosis and gap-junctional-intercellular communication (GJIC). GJIC, the movement of small molecules (less than 1.2 kD) through membrane channels, is important in regulating cellular homeostasis and differentiation. The inhibition of hepatic GJIC can increase cell proliferation and possibly, inhibit apoptosis. In this study, the relationship between hepatic GJIC, proliferation, and apoptosis was examined in rats treated for 7 days with tumor-promoting doses of the nongenotoxic hepatocarcinogens phenobarbital (PB; 800 ppm), pregnenolone-16alpha-carbonitrile (PCN; 1000 ppm), and Aroclor 1254 (PCB; 100 ppm). In addition, 3-methylcholanthrene (3MC) was included as a negative control. PB, PCN, and PCB increased parenchymal-cell proliferation and inhibited hepatic apoptosis, while no alteration in these growth parameters was observed in 3MC-treated rats. GJIC, as measured by fluorescent-dye transfer through intact liver, was decreased nearly 50% by PB, PCN, and PCB, yet no effect on GJIC was observed in liver from 3MC-treated rats. These data indicate that compounds that inhibit GJIC in liver may be nongenotoxic hepatocarcinogens, which occurs simultaneously during increased cell proliferation and inhibited apoptosis.

The UDP-glucuronyltransferase inducers, phenobarbital and pregnenolone-16alpha-carbonitrile, enhance thyroid-follicular cell apoptosis: association with TGF-beta1 expression.

Exposure to certain UDP-glucuronosyltransferase (UDP-GT) inducers leads to follicular cell hyperplasia, and ultimately thyroid gland tumors. These compounds decrease thyroid hormones, which increases serum concentrations of thyroid stimulating hormone (TSH). This induction of TSH enhances thyroid-follicular cell proliferation. In addition, treatment with classical goitrogenic compounds, such as propylthiouracil (PTU) and methimazole (MMI), induces TGF-beta1 in thyroid-follicular cells, presumably through increased TSH. In other tissues, increases in TGF-beta1 induce apoptosis, a particular form of programmed cell death. In this experiment, we sought to determine whether the UDP-GT inducers, phenobarbital (PB) and pregnenolone-16alpha-carbonitrile (PCN) modulate thyroid-follicular cell apoptosis. If so, are the induction of apoptosis and TGF-beta1 possibly linked? An additional group of rats treated with the thyroid goitrogen, PTU was included. Male Sprague-Dawley rats were treated with thyroid hormone disrupting doses of PB, PCN, or PTU for 3, 7, 14, 21, 28, 45, or 90 days. In this study, PTU treatment increased apoptosis and TGF-beta1 immunoreactive thyroid-follicular cells. PTU treatment of rats produced both a large increase number of TGF-beta1-positive cells (detected by immunohistochemistry), and apoptotic thyroid-follicular cells (detected by morphology). In PB- and PCN-treated rats, a moderate increase in apoptosis coincided with similar increases in TGF-beta1 immunoreactive thyroid-follicular cells. In summary, PB and PCN increase apoptosis and the percentage of TGF-beta1 positive thyroid-follicular cells. Thus, treatment with UDP-GT-inducing chemicals may increase the expression of TGF-beta1 and apoptosis in the thyroid to compensate for the thyroid hypertrophy and hyperplasia.

Effect of dietary antioxidants on dieldrin-induced hepatotoxicity in mice.

An increasing number of reports suggest that oxidative stress plays a role in the toxicity of various xenobiotics, including organochlorine pesticides and drugs such as phenobarbital. Antioxidants appear to be protective against the damage induced by an acute dose of endrin, supporting the theory of a role for reactive oxygen in the toxicity of this class of compounds. The current study examined the effects of the dietary administration of vitamin C (400 mg/kg diet) or vitamin E (200 mg DL-alpha-tocopherol acetate/kg diet) on hepatotoxicity induced by subchronic (7 or 28 days) feeding of dieldrin (1, 3 and 10 mg/kg diet) to male B6C3F1 mice. Hepatoxicity induced by feeding of dieldrin for 28 days was evidenced by liver enlargement, hypertrophy of centrolobular hepatocytes, induction of hepatic ethoxyresorufin O-deethylase activity, and increased DNA synthesis in hepatocytes, particularly in centrolobular hepatocytes. Neither vitamin inhibited the dose-dependent increase in liver/body weight ratios, hypertrophy of centrolobular hepatocytes, or induction of hepatic ethoxyresorufin O-deethylase. Vitamin E, however, inhibited hepatic DNA synthesis at all dietary intakes of dieldrin, while vitamin C was inhibitory at 1 and 3, but stimulatory at 10 mg dieldrin per kg diet. The major changes in DNA labeling occurred in the centrolobular zones, but were not consistently inhibited by vitamins C or E. The ability of antioxidant vitamins to inhibit dieldrin-induced hepatic DNA synthesis suggests oxidative stress is involved in the toxicity of this compound; however, the inability of these vitamins to prevent all hepatotoxic changes indicates other factors are also involved.

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.

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.

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.

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.

Investigation of mechanism of drug-induced cardiac injury and torsades de pointes in cynomolgus monkeys.

BACKGROUND AND PURPOSE: Drug candidates must be thoroughly investigated for their potential cardiac side effects. During the course of routine toxicological assessment, the compound RO5657, a CCR5 antagonist, was discovered to have the rare liability of inducing torsades de pointes (polymorphic ventricular arrhythmia) in normal, healthy animals. Studies were conducted to determine the molecular mechanism of this arrhythmia. EXPERIMENTAL APPROACH: Toxicological effects of repeat dosing were assessed in naïve monkeys. Cardiovascular effects were determined in conscious telemetry-implanted monkeys (repeat dosing) and anaesthetized instrumented dogs (single doses). Mechanistic studies were performed in guinea-pig isolated hearts and in cells recombinantly expressing human cardiac channels. KEY RESULTS: In cynomolgus monkeys, RO5657 caused a low incidence of myocardial degeneration and a greater incidence of ECG abnormalities including prolonged QT/QTc intervals, QRS complex widening and supraventricular tachycardia. In telemetry-implanted monkeys, RO5657 induced arrhythmias, including torsades de pointes and in one instance, degeneration to fatal ventricular fibrillation. RO5657 also depressed both heart rate (HR) and blood pressure (BP), with no histological evidence of myocardial degeneration. In the anaesthetized dog and guinea-pig isolated heart studies, RO5657 induced similar cardiovascular effects. RO5657 also inhibited Kv11.1 and sodium channel currents. CONCLUSIONS AND IMPLICATIONS: The molecular mechanism of RO5657 is hypothesized to be due to inhibition of cardiac sodium and Kv11.1 potassium channels. These results indicate that RO5657 is arrhythymogenic due to decreased haemodynamic function (HR/BP), decreased conduction and inhibition of multiple cardiac channels, which precede and are probably the causative factors in the observed myocardial degeneration.

Vitamin E modulation of hepatic focal lesion growth in mice.

The effect of DL-alpha-tocopherol acetate (vitamin E) on 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 dose groups: 0 mg vitamin E/kg NIH-07 diet, 50 mg vitamin E/kg NIH-07 diet (control diet), 250 mg vitamin E/kg NIH-07 diet, and 450 mg vitamin E/kg NIH-07 diet. Mice were euthanized after either 30 or 60 days of dietary treatment. In normal (nonlesion) liver, vitamin E deficiency (0 mg/kg diet) increased hepatic DNA synthesis. In addition, vitamin E supplementation (450 mg/kg diet) decreased the incidence of hepatic apoptosis, while vitamin E deficiency (0 mg/kg diet) increased the incidence of hepatic apoptosis. The effect of vitamin E-induced lesion growth was examined by measuring the number of focal lesions per liver and the relative focal lesion volume. High-dose vitamin E supplementation (450 mg/kg diet) appeared to enhance the growth of hepatic focal lesions. In particular, basophilic lesions appeared to be the most sensitive to high-dose vitamin E modulation (450 mg/kg diet) as evidenced by increased number, volume, and labeling index of hepatic focal lesions. Vitamin E deficiency also appeared to enhance the growth of hepatic focal lesions, though to a lesser extent than vitamin E supplementation (450 mg/kg diet). In the present study, both vitamin E supplementation (450 mg/kg diet) and deficiency (0 mg/kg diet) appeared to enhance focal lesion growth albeit neither treatment enhanced lesion growth as dramatically as known nongenotoxic hepatocarcinogens (e.g., phenobarbital and dieldrin). The data presented here suggest that oxidative stress in focal hepatocytes may be a component of the liver tumor promotion process.

Inhibition of tumor promotion and hepatocellular growth by dietary restriction in mice.

The effects of dietary restriction on the growth of hepatic focal lesions in phenobarbital (PB) promoted mice were examined. Dietary restriction which can inhibit many age-related diseases in rodents including hepatic cancer also decreases cell proliferation and increases apoptosis in the liver. In contrast, PB, a non-genotoxic rodent hepatocarcinogen, enhances the growth of hepatic focal lesions in mice and rats by increasing cell proliferation and inhibiting apoptosis. The present study examined the impact of dietary restriction on PB-induced hepatic tumor promotion. Focal lesions were produced by diethylnitrosamine (DEN) treatment (35 mg DEN/kg body weight injections, twice per week for 8 weeks). After lesions were produced, mice were placed into one of the following four groups: NIH-07 control diet/no PB (group 1); NIH-07 diet/500 mg PB per liter of drinking water (group 2); dietary restricted NIH-07 diet/no PB (group 3); and dietary restricted NIH-7 diet/ 500 mg PB per liter of drinking water (group 4). In this study, PB (500 mg/l) treatment to ad libitum-fed mice (group 2) enhanced focal lesion volume, number, and labeling index compared with group 1. In addition, PB treatment (group 2) inhibited apoptosis in normal and focal hepatocytes compared with untreated control mice (group 1). In contrast, in dietary restricted mice treated with PB (group 4) a significantly lower focal lesion volume, number and labeling index were seen compared with the ad libitum-fed/PB treatment group (group 2). PB treatment in dietary restricted mice (group 4) did not inhibit focal apoptosis, in fact, the incidence of focal apoptosis was increased in these mice compared with ad libitum and PB-treated mice (group 2). In dietary restricted mice treated with PB (group 4), the ability of PB to promote the growth of preneoplastic focal lesions was inhibited. These results show that dietary restriction can ablate the tumor promotional effects of PB in hepatic focal lesions and suggest that inhibition of focal lesion DNA synthesis and enhancement of apoptosis may be a mechanism for this effect.

Effect of partial hepatectomy on the expression of seven rat sulphotransferase mRNAs.

1. The effect of partial hepatectomy on the expression of sulphotransferase (SULT) mRNA was studied. SULTs fall into two distinct classes based on substrate preference: phenol SULT1 family (SULT1A1, SULT1B1, SULT1C1 and SULT1E2) and hydroxysteroid SULT2 family (SULT20/21, SULT40/41 and SULT60). 2. Hepatic expression of SULT mRNA was analysed in the sham-hepatectomised rat (sham) and in the partially hepatectomised (PH) rat at various times after PH. Northern-blot analysis with [alpha-32P]dATP-labelled oligonucleotide probes specific for individual SULT mRNAs was used to monitor hepatic SULT mRNA expression. In general, SULT mRNAs underwent a decrease in expression after PH and the magnitude of decrease was dependent on the SULT isoform. 3. The decrease in SULT mRNA expression was resolved and even induced (SULT40/41 in the female rat) by 10-30 days after PH. Of the phenol SULT isoforms, both SULT1C1 and SULT1E2 mRNAs were significantly decreased by 18-24 h after PH in the male rat. The other phenol SULTs (SULT1A1 and SULT1B1) tended to decrease in the male rat after PH, but the decreases were not statistically significant. Expression of SULT20/21 mRNA was decreased in the female rat (80% at 24 h) and fully recovered by 10 days. SULT40/41 mRNA tended to decrease after PH; however, the decrease was not statistically significant. SULT 60 mRNA was decreased from 24 to 96 h after PH. 4. Thus, during the period of rapid liver growth that occurs after partial hepatectomy, SULT mRNA expression is decreased. The phenomenon of decreased SULT mRNA expression is similar to other states of rapid liver growth (e.g. cancer tissue and young animals) in which expression of SULT enzymes is characteristically low.

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.

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.

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.