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.

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.

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.