The effect of metformin in EML4-ALK+ lung cancer alone and in combination with crizotinib in cell and rodent models.

Cell based studies have suggested that the diabetes drug metformin may combine with the anaplastic lymphoma kinase receptor (ALK) inhibitor crizotinib to increase ALK positive lung cancer cell killing and overcome crizotinib resistance. We therefore tested metformin alone and in combination with crizotinib in vivo, by employing a xenograft mouse model of ALK positive lung cancer. We found that 14 days of daily oral metformin (100 mg/kg) alone had a moderate but statistically significant effect on tumour growth suppression, but in combination with crizotinib, produced no greater tumour suppression than crizotinib (25 mg/kg) alone. We also reassessed the effect of metformin on EML4-ALK positive lung cancer (H3122) cell viability. Although metformin alone did have a moderate effect on cell viability (30% suppression) this was only at a clinically irrelevant concentration (5 mM) and there was no additive effect with cytotoxic concentrations of crizotinib. Moreover, metformin did not overcome crizotinib resistance in our resistant cells. Nevertheless, we were able to show that metformin induces a G1-cell cycle arrest and apoptosis alone and in combination with crizotinib. Also, consistent with earlier work, the addition of insulin-like growth factor-1 (IGF-1) to EML4-ALK positive cancer cells reduced cell killing by crizotinib. We therefore hypothesised that the effect of metformin in vivo was not due to direct cytotoxicity on cancer cells, but by modulation of IGF-1 expression. We therefore measured levels of IGF-1 in plasma taken from mice treated with metformin, but found no difference between the drug treatment and control groups. We further hypothesised that the effect of metformin could be due to modulation of thrombospondin 1 (TSP-1), which metformin has been proposed to regulatein vivo, but again we found no difference between the experimental groups. Finally, we investigated the potential for liver and kidney toxicity, as well as CYP3A based interactions, from the combination of metformin with crizotinib.

Inhibition of Mitogen-Activated Protein Kinase Kinase Alone and in Combination with Anaplastic Lymphoma Kinase (ALK) Inhibition Suppresses Tumor Growth in a Mouse Model of ALK-Positive Lung Cancer.

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer most commonly arises through EML4 (Echinoderm Microtuble Like 4)-ALK chromosomal fusion. We have previously demonstrated that combination of the ALK inhibitor crizotinib with the MEK inhibitor selumetinib was highly effective at reducing cell viability of ALK-positive non-small-cell lung cancer (H3122) cells. In this study, we further investigated the efficacy of crizotinib and selumetinib combination therapy in an in vivo xenograft model of ALK-positive lung cancer. Crizotinib decreased tumor volume by 52% compared with control, and the drug combination reduced tumor growth compared with crizotinib. In addition, MEK inhibition alone reduced tumor growth by 59% compared with control. Crizotinib and selumetinib alone and in combination were nontoxic at the dose of 25 mg/kg, with values for ALT (<80 U/l) and creatinine (<2 mg/dl) within the normal range. Our results support the combined use of crizotinib with selumetinib in ALK-positive lung cancer but raise the possibility that a sufficient dose of an MEK inhibitor alone may be as effective as adding an MEK inhibitor to an ALK inhibitor. SIGNIFICANCE STATEMENT: This study contains in vivo evidence supporting the use of combination MEK inhibitors in ALK+ lung cancer research, both singularly and in combination with ALK inhibitors. Contrary to previously published reports, our results suggest that it is possible to gain much of the benefit from combination treatment with an MEK inhibitor alone, at a tolerable dose.

Mechanisms of suppression of cell growth by dual inhibition of ALK and MEK in ALK-positive non-small cell lung cancer.

Anaplastic lymphoma kinase (ALK) rearrangement, a key oncogenic driver in a small subset of non-small cell lung cancers, confers sensitivity to ALK tyrosine kinase inhibitors (TKIs). Crizotinib, a first generation ALK-TKI, has superiority to standard chemotherapy with longer progression-free survival and higher objective response rate. However, clinical benefit is limited by development of resistance, typically within a year of therapy. In this study the combined effect of crizotinib and the MEK inhibitor selumetinib was investigated in both crizotinib naïve (H3122) and crizotinib resistant (CR-H3122) ALK-positive lung cancer cells. Results showed that combination treatment potently inhibited the growth of both H3122 and CR-H3122 cells, resulting from increased apoptosis and decreased cell proliferation as a consequence of suppressed downstream RAS/MAPK signalling. The drug combination also elicited a greater than 3-fold increase in Bim, a mediator of apoptosis, and p27, a cyclin dependent kinase inhibitor compared to crizotinib alone. The results support the hypothesis that combining MEK inhibitors with ALK inhibitor can overcome ALK inhibitor resistance, and identifies Bim, PARP and CDK1 as druggable targets for possible triple drug therapy.

Deer velvet supplementation decreases the grade and metastasis of azoxymethane-induced colon cancer in the male rat.

Since deer velvet (DV) extract promotes angiogenesis, its ability to modulate the growth and invasiveness of colon tumours was investigated. Male Wistar rats were each given a subcutaneous injection of azoxymethane (AOM) at 15 mg/kg once a week for 3 weeks. One week following the last dose of AOM the rats received either 1g/kg of DV delivered in a cube of raspberry gelatin or plain raspberry gelatin daily for 26 weeks. At necropsy, tumours were measured and the distance from the anus was recorded. Tissue samples were categorised according to the Astler-Coller system. The results showed that there were no significant differences in most parameters examined (i.e. body weight gain, multiplicity, tumour volume and incidence). The only statistically significant differences seen were associated with metastasis and tumour grade. Specifically, more of the tumours in the DV-treated rats were of a lower grade compared to the controls, both when all tumour sites were considered (0.91 vs. 0.66, p<0.0001), as well as those located only in the colon (0.95 vs. 0.84, p<0.03). Therefore, this study can confidently conclude that DV does not increase the incidence, multiplicity, metastasis or tumour volume of AOM-induced colon cancer in the rat.

A new role for tamoxifen in oestrogen receptor-negative breast cancer when it is combined with epigallocatechin gallate.

We have previously shown that tamoxifen+epigallocatechin gallate (EGCG) is synergistically cytotoxic towards oestrogen receptor (ER)-negative breast cancer cells. To determine if this response would correlate with significant tumour suppression in vivo, athymic nude female mice were implanted with MDA-MB-231 cells and treated with tamoxifen, EGCG, EGCG+tamoxifen, or vehicle control for 10 weeks. Tumour volume in EGCG- (25 mg kg(-1))+tamoxifen (75 microg kg(-1))-treated mice decreased by 71% as compared with vehicle control (P<0.05), whereas tumour weight was decreased by 80% compared with control (P<0.01). Epigallocatechin gallate treatment did not alter ER protein expression in MDA-MB-231 cells and thus was not a mechanism for the observed tumour suppression. However, western blotting of tumour extracts demonstrated that epidermal growth factor receptor (EGFR; 85% lower than control), pEGFR (78% lower than control), mammalian target of rapamycin (mTOR; 78% lower than control), and CYP1B1 (75% lower than control) were significantly lower after the combination treatment as compared with all other treatments. Nuclear factor-kappaB (NF-kappaB), b-Raf, p-MEK, S6K, 4EBP1, Akt, vascular EGFR-1 (VEGFR-1) and VEGF expressions were decreased in control but not in the individual treatments, whereas MEK, phospholipase D 1/2, TGF alpha, and ERK expressions were not changed after any treatment. The results demonstrate that tamoxifen at realistic doses (75 mug kg(-1)) can suppress the growth of ER-negative breast cancer when combined with EGCG. In addition, the dominant mechanism for tumour suppression is the concomitant decrease in tumour protein expressions of mTOR and the EGFR.

Sex- and strain-dependent effects of epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) in the mouse.

We have previously demonstrated that 50mg/kg of epigallocatechin gallate (EGCG) is hepatotoxic to female Swiss Webster mice, while lower doses of EGCG and epicatechin gallate (ECG) modulate various cytochrome P450 (CYP) isoforms. Therefore, this study was designed to further investigate the role of strain and sex in catechin-mediated enzyme modulation and hepatotoxicity in mice. Male and female BALB/c and male Swiss Webster mice were treated with either ECG or EGCG (25 and 50 mg/kg, ip) for 7 days. The results demonstrated that EGCG (50 mg/kg) produced severe hepatic necrosis, elevated ALT activities and a 20% mortality rate in male Swiss Webster mice and mild hepatotoxicity in male BALB/c mice. In female BALB/c mice the mortality rate was 20%, which correlated with extensive hepatic necrosis. Of the two catechins, only ECG significantly inhibited CYP isoforms. Specifically, prostatic aromatase activity decreased by 31+/-2%, while CYP1A catalytic activity and polypeptide levels were decreased 29+/-6% and 25+/-4%, respectively. However, CYP2E1 and CYP3A activity remained unchanged following ECG administration. Additionally, EGCG did not alter aromatase, CYP1A, CYP3A or CYP2E1 in male Swiss Webster mice. In conclusion, EGCG (50 mg/kg) elicits mortality in both male and female Swiss Webster mice, as well as female BALB/c mice. ECG significantly inhibits both aromatase and CYP1A in male Swiss Webster mice. Therefore, sex-specific modulation of CYP isoforms occurs following administration of EGCG and ECG in Swiss Webster mice.

Tamoxifen and epigallocatechin gallate are synergistically cytotoxic to MDA-MB-231 human breast cancer cells.

High concentrations of specific catechins [epigallocatechin gallate (EGCG), epigallocatechin (EGC) and epicatechin gallate (ECG)] inhibit the proliferation of many different cancer cell lines. The aim of this work was to determine if low concentrations of catechins with and without 4-hydroxytamoxifen (4-OHT) co-treatment would cause significant cytotoxicity in estrogen receptor-positive (ERalpha+) and -negative (ERalpha-) human breast cancer cells. Therefore, MCF-7, T47D, MDA-MB-231 and HS578T cells were incubated with EGCG, EGC or ECG (5-25 microM) individually and in combination with 4-OHT for 7 days. Cell number was determined by the sulforhodamine B cell proliferation assay. As single agents, none of the catechins were cytotoxic to T47D cells, while only EGCG (20 microM) elicited cytotoxicity in MCF-7 cells. Additionally, no benefit was gained by combination treatment with 4-OHT. ERalpha- human breast cancer cells were more susceptible as all three catechins were significantly cytotoxic to HS578T cells at concentrations of 10 microM. In this cell line, combination with 4-OHT did not increase cytotoxicity. However, the most striking results were produced in MDA-MB-231 cells. In this cell line, EGCG (25 microM) produced a greater cytotoxic effect than 4-OHT (1 microM) and the combination of the two resulted in synergistic cytotoxicity. In conclusion, low concentrations of catechins are cytotoxic to ERalpha- human breast cancer cells, and the combination of EGCG and 4-OHT elicits synergistic cytotoxicity in MDA-MB-231 cells.

Estrogen receptor-mediated actions of polyphenolic catechins in vivo and in vitro.

Recent investigations have demonstrated that polyphenolic catechins inhibit breast cancer cell proliferation and tumor growth. However, the ER-mediated effects of the three predominant catechins (EGCG, ECG, and EGC) have not been extensively examined in vitro or in vivo. Therefore, EGCG, ECG, and EGC were examined for their ability to compete with [(3)H]-17beta-estradiol ([(3)H]-E(2)) for binding to ERalpha and ERbeta and to elicit reporter gene activity in MCF-7 human breast cancer cells transiently transfected with either chimeric ERalpha or ERbeta. EGCG and ECG displaced [(3)H]-E(2) from GST-hERalphadef (D, E, and F domains of human ERalpha fused to GST) or from full-length human ERbeta. Additionally, only EGCG elicited Gal4-hERalphadef and Gal4-mERbetadef-mediated reporter gene expression (EC(50) values: 28 and 19 micro M, respectively) in MCF-7 cells cotransfected with a Gal4-regulated luciferase reporter gene. In cotreatment experiments, EGCG (1-50 micro M) and ECG (1 micro M) decreased E(2)-induced (1 nM) ERbeta-mediated gene expression 35-50%. In vivo, no catechin induced estrogenic responses (uterine weight or uterine peroxidase activity) in immature C57BL/6 mice. However, when mice were cotreated with E(2) (10 micro g/kg/day, 3 days) and either EGCG (30 and 50 mg/kg/day, 3 days) or ECG (50 mg/kg/day, 3 days), uterine peroxidase activity was increased 2.3-fold above that elicited by E(2) alone. In conclusion, EGCG and ECG bind to ERalpha and ERbeta, but only EGCG elicited ER-mediated gene expression in vitro. However, both of these compounds moderately increased E(2)-inducible responses in vivo.

Methylphenidate inhibits cytochrome P450 in the Swiss Webster mouse.

Drug interactions have previously been reported following the co-administration of methylphenidate (MPH) and drugs metabolized by the cytochrome P450 (CYP450) system such as imipramine. Therefore, this study used the Swiss Webster mouse to determine the effect of MPH on CYP450 isozymes likely to be important in the interaction between MPH and imipramine. Single high doses of MPH (25, 50 and 100 mg/kg, i.p.) were administered to simulate the abuse of MPH. Under these conditions, MPH decreased total hepatic CYP450 to 50% of control. Additionally, MPH inhibited the catalytic activity of CYP1A and CYP2E1 by 50%, and decreased the polypeptide levels of CYP3A by 30%. In a second study designed to simulate more closely therapeutic use, MPH was administered orally for two weeks at 10-fold lower doses (2.5, 5 and 10 mg/kg/day). MPH decreased total hepatic CYP450 at both 5 and 10 mg/ kg/day (0.96 +/- 0.01 and 0.96 +/- 0.06 nmol/mg versus 1.34 +/- 0.01 nmol/mg for saline control, P<0.05). The catalytic activity and protein levels of CYP1A were diminished by up to 50% of control, while catalytic activity and polypeptide levels for CYP2E1 and CYP3A remained unchanged. These results indicate that MPH inhibits the CYP450 system following both abuse and therapeutic scenarios. However, this effect was dependent on both the isoform of CYP450 and the duration of MPH administration.

Short term treatment with St. John's wort, hypericin or hyperforin fails to induce CYP450 isoforms in the Swiss Webster mouse.

This investigation was designed to determine whether St. John's wort (SJW)(435 mg/kg/d), a readily available antidepressant, or its purported active constituents hypericin (1 mg/kg/d) and hyperforin (10 mg/kg/d) were able to induce various hepatic cytochrome P450 (CYP450) isoforms. SJW, hypericin and hyperforin were administered to male Swiss Webster mice for four consecutive days and hepatic microsomes were prepared on day 5. None of the three treatments resulted in a statistical change in total hepatic CYP450 (SJW treated 0.95 +/- 0.09 nmol/mg vs control 1.09 +/- 0.14 nmol/mg). Furthermore, the catalytic activities of CYP1A2. CYP2E1 and CYP3A were unchanged from control following all three treatments as determined by ethoxyresorufin O-deethylation, p-nitrophenol hydroxylation and erythromycin N-demethylation respectively. Additionally, western immunoblotting demonstrated that there was no significant change in the polypeptide levels of any of the three isoforms. These results indicate that four days of treatment with moderate to high doses of SJW, hyperforin or hypericin fails to induce these CYP450 isoforms in the male Swiss Webster mouse.

St. John's wort extract induces CYP3A and CYP2E1 in the Swiss Webster mouse.

This investigation was designed to determine the ability of St. John's wort (SJW), a readily available antidepressant, to induce various hepatic drug metabolizing enzymes. SJW (140 or 280 mg/kg/day) was administered to male Swiss Webster mice for 1, 2, or 3 weeks. Enzymatic activity was analyzed in hepatic microsomes for all of the following drug metabolizing enzymes: CYP3A, CYP1A, CYP2E1, and UDP-glucuronosyltransferase (UDPGT). The catalytic activity of CYP1A was unchanged from control following any dose or duration of SJW, while both CYP3A and CYP2E1 catalytic activities were increased 2-fold by both SJW concentrations but only following 3 weeks of administration. Results from Western immunoblotting studies supported the changes in catalytic activity, as protein levels for CYP2E1 and CYP3A were increased (2.5-fold and 6-fold, respectively) following 3 weeks of SJW administration. Additionally, the catalytic activity of the conjugation enzyme UDPGT was unchanged from control following all SJW treatments. These results indicate that in the mouse moderate doses of SJW cause an increase in the catalytic activity and polypeptide levels of CYP2E1 and CYP3A but only following 21 days of administration, while the catalytic activity of CYP1A and UDPGT activity remain unaffected.

Retinol potentiates acetaminophen-induced hepatotoxicity in the mouse: mechanistic studies.

This study was designed to elucidate the mechanism of retinol's potentiation of acetaminophen-induced hepatotoxicity. To accomplish this, the major bioactivation and detoxification pathways for acetaminophen were investigated following retinol (75 mg/kg/day, 4 days), acetaminophen (400 mg/kg), and retinol + acetaminophen treatment. Hepatic microsomes were used to determine the catalytic activity and polypeptide levels of cytochrome P450 enzymes involved in the murine metabolism of acetaminophen. Results showed that the catalytic activity and polypeptide levels of CYP1A2, CYP2E1, and CYP3A were unchanged in the treatment groups compared to vehicle and untreated controls. In combination, retinol + acetaminophen caused a significantly greater depletion of GSH compared to corn oil + acetaminophen (0.36 +/- 0.11 vs 0.89 +/- 0.19 micromol/g, respectively, p < 0.05). This greater GSH depletion correlated with a higher degree of hepatic injury in the retinol + acetaminophen-treated animals but is probably not the cause of the potentiated injury since the results showed that retinol treatment itself did not alter hepatic glutathione (3.34 +/- 0.43 vs 3.44 +/- 0.46 micromol/g for retinol vs vehicle, respectively). However, hepatic UDPGA stores were decreased in the retinol-treated group compared to untreated and corn oil controls (54.6 +/- 10.6 vs 200.6 +/- 17.6 nmol/g for retinol and untreated control, respectively, p < 0.001). This demonstrates that there is significantly less hepatic UDPGA available for conjugation following retinol administration. The results suggest that decreased hepatic UDPGA is likely the cause of retinol's potentiation of acetaminophen-induced hepatic injury.

Acetaminophen elicits anti-estrogenic but not estrogenic responses in the immature mouse.

Acetaminophen is a widely used analgesic, which has exhibited evidence of estrogenic activity in estrogen-receptor positive human breast cancer cells. However, there is limited in vivo experimentation regarding the estrogenicity of acetaminophen. Therefore, the present study examined the in vivo estrogenic potential of acetaminophen using the immature female mouse model. Specifically, C57Bl/6 female mice were treated with acetaminophen (100, 200 and 250 mg/kg per day, i.p.) for 3 consecutive days. Positive controls received estradiol (10 microg/kg per day, i.p.) for 3 consecutive days. Markers of estrogenic activity examined were uterine weight, uterine peroxidase activity and progesterone receptor (PR) up-regulation. The results demonstrated that, at all three doses, acetaminophen did not significantly increase uterine weight, uterine peroxidase activity or PR levels. However, the co-administration of E(2) and acetaminophen (200 mg/kg per day, 3 days, i.p.) resulted in a decrease in the E(2)-induced upregulation of uterine and hepatic nuclear PR (uterine PR values of 39.7+/-6.6 and 23.7+/-3.4 fmol/mg for E(2)+vehicle and E(2)+acetaminophen, respectively, P<0.05). Additionally, the estradiol-induced increase in uterine peroxidase was decreased 75% by acetaminophen (200 mg/kg per day, 3 days, i.p.). Therefore, in the immature mouse model acetaminophen treatment did not elicit estrogenic activity. However, acetaminophen had a limited ability to antagonize the effects of E(2).

The effect of retinol on hepatic and renal drug-metabolising enzymes.

Retinol pretreatment (75 mg/kg/day, 4 days) potentiated paracetamol-induced hepatotoxicity in BALB/c mice (alanine aminotransferase (ALT) activity; 2510+/-602 vs 1155+/-282 IU/l; retinol+paracetamol vs corn oil+paracetamol, respectively, P<0.05); however, this potentiation did not occur in the kidney, indicating an organ-specific response. Retinol treatment alone was not toxic in either organ, as indicated by ALT activity, blood urea nitrogen and creatinine. The potentiation effect could be mediated by retinol's induction of CYP450 isoforms relevant to paracetamol metabolism or through depletion of glutathione. Therefore, these parameters were investigated in both organs. Following retinol treatment, renal CYP2E1 and hepatic CYP1A2 and CYP2E1 catalytic activities and polypeptide levels were unchanged. However, retinol significantly decreased both the catalytic activity (0.23+/-0.03 vs 0.53+/-0.06 nmol/mg/min; retinol vs untreated, respectively, P<0.05) and polypeptide levels (58+/-0.6% of control) of hepatic CYP3A. Inhibition of renal CYP3A did not occur as catalytic activity and polypeptide levels were unchanged from control. Following retinol treatment, total reduced glutathione levels, in both organs, were not significantly different from control. Therefore, the potentiation of paracetamol-induced hepatotoxicity is independent of CYP450 and glutathione.

Role of cytochrome P4502E1 in retinol's attenuation of carbon tetrachloride-induced hepatotoxicity in the Swiss Webster mouse.

In the mouse, retinol administration attenuates carbon tetrachloride (CCl4)-induced hepatic injury. We have investigated the role of cytochrome P4502E1 (CYP2E1) in this interaction. Male Swiss Webster mice were administered retinol (75 mg/kg/d) or vehicle for 3 days prior to CCl4 (30 microl/kg, ip). Hepatotoxicity produced by CCl4 was assessed by plasma alanine aminotransferase (ALT) activity and light microscopy (ALT activity of 1391+/-430 vs. 274+/-92 IU/L for vehicle + CCl4 and retinol + CCl4 treatments respectively, p < 0.05). Retinol's attenuation of liver injury was maintained when CCl4 was administered 48 h after the conclusion of the retinol pretreatment. Aniline hydroxylation activity, an indicator of CYP2E1 catalytic activity, determined on day 4 was 33.8% of untreated control in vehicle + CCl4 treatments while the retinol + CCl4 treatment group was 94.2% of untreated control. Additionally, CYP2E1 immunoreactive protein was 78% lower in vehicle + CCl4 vs. retinol + CCl4 treatment groups. Attenuation of potentiated hepatotoxicity was also observed when CYP2E1 was induced by acetone (ALT activity of 3119+/-1066 vs. 247+/-77 IU/L for vehicle and retinol treatments respectively, p < 0.05). In the mouse, retinol itself does not alter constitutive or inducible CYP2E1 expression. However, in combination with CCl4 retinol does reduce the amount of CCl4 bioactivated to its toxic metabolite. We conclude that retinol attenuates CCl4-induced hepatotoxicity by causing a decrease in CCl4 bioactivation but does not cause a decrease in CYP2E1 expression.

The interactions between retinol and five different hepatotoxicants in the Swiss Webster mouse.

The interactive effects between retinol and various hepatotoxicants (allyl alcohol, acetaminophen, carbon tetrachloride, D-galactosamine, and phalloidin) were studied in the male Swiss Webster mouse. The mice were administered retinol at 75 mg/kg/day (or the vehicle of retinol) by oral gavage for 7 days. Hepatoxicity produced by the chemicals was determined by plasma alanine aminotransferase (ALT) activity and histopathology. After 7 days of retinol pretreatment, the hepatotoxicities of allyl alcohol, acetaminophen, and galactosamine were potentiated. Interestingly, the hepatotoxicity of carbon tetrachloride and phalloidin was protected by identical retinol pretreatment. Microscopic examination of histologic liver sections demonstrated the specific hepatic necrosis associated with each individual chemical and confirmed the ALT values obtained. Once an interaction between retinol and the five hepatotoxicants was established, the duration of retinol pretreatment necessary to elicit an interaction was determined for each hepatotoxicant. Results demonstrated that the duration of retinol pretreatment was specific for each hepatotoxicant. The accumulation of retinoids in the liver during retinol pretreatment was determined using high-performance liquid chromatography analysis. Significant increases in the basal liver levels of retinol and retinyl palmitate were seen within 1 to 3 days of retinol treatment compared to control. Retinol pretreatment resulted in potentiation or protection of specific hepatotoxicant-induced liver damage. Currently, studies are being conducted which probe into the mechanisms of these interactions.

Vitamin A modulation of xenobiotic-induced hepatotoxicity in rodents.

Vitamin A (VA, retinol) has been shown to modulate cells of the immune system. When rats are pretreated with VA (75 mg/kg/day) for 7 days, there is greatly potentiated liver damage upon subsequent exposure to hepatotoxicants such as CCl4. This potentiated damage can be blocked by superoxide dismutase or catalase, suggesting that reactive oxygen species are playing a major role in the increased liver injury. The studies reported here examined VA-induced modulation of CCl4 hepatotoxicity in different strains of male rats, female rats, and different strains of male mice. Also, the role of VA-induced weight loss on potentiation of CCl4 injury was investigated. Rats or mice were dosed with VA (retinol) at 75 mg/kg/day, po, for 7 days. In an additional VA dose-response study, mice were given VA at 18.8, 37.5, or 75 mg/kg/day, po, for 7 days. On day 8 they were given a dose of CCl4 which elicited mild hepatic damage. On day 9 they were necropsied. Male and female Sprague-Dawley rats, and male Fischer-344 and athymic nude rats pretreated with VA had an approximately 10-fold increase in liver damage as compared to vehicle controls. Pretreatment of male Balb/C, C3H/HeJ, Swiss-Webster, or athymic nude mice resulted in a marked reduction of CCl4-induced hepatic damage. In the dose-response study in mice, increasing doses of VA elicited increasing amounts of protection from CCl4-induced liver injury. Paired feeding studies revealed that VA-induced weight loss (or decreased weight gain) had no effect on subsequent VA-induced potentiation (rats) or protection (mice) from hepatic damage caused by CCl4.(ABSTRACT TRUNCATED AT 250 WORDS)