Cytotoxicity of propyl gallate and related compounds in rat hepatocytes

 The cytotoxic effects of propyl gallate (PG), its related gallates and gallic acid have been studied in freshly isolated rat hepatocytes. Addition of PG (0.5–2.0 mM) to hepatocyte suspension elicited concentration-dependent cell death accompanied by losses of intracellular ATP, adenine nucleotide pools, glutathione (GSH) and protein thiols. The rapid loss of intracellular ATP preceded the onset of cell death caused by PG. In the comparative toxic effects of PG and related gallates at concentration of 1 mM, octyl gallate (OG), dodecyl gallate (DG) and butyl gallate (BG) elicited an abrupt depletion of ATP, followed by an acute cell death. These gallates were more toxic than PG; the toxic effects of PG were similar to those of methyl gallate (MG) and ethyl gallate (EG). In mitochondria isolated from rat liver, PG caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by OG and DG. According to the respiratory control index, the order of impairment potency to mitochondria was OG>BG, DG>PG>EG, MG>gallic acid. These results indicate that PG and related gallates are toxic to hepatocytes and that the acute cytotoxicity may be due to mitochondrial dysfunction. Received: 16 May 1994 / Accepted: 15 August 1994     

Propyl gallate-induced DNA fragmentation in isolated rat hepatocytes

Incubation of isolated rat hepatocytes with propyl gallate (PG) at concentrations of ≥1 mM induced cell killing, whereas PG at ≤0.5 mM did not cause cell death during a 3-h incubation. PG at ≥0.5 mM elicited the ladder formation of soluble low-molecular weight DNA fragments with integer multiples of approximately 180 bp and specific nuclear DNA cleavages detected cytopathologically by labeling of a digoxigenin-nucleotide complex to new 3′-OH ends. Both of these PG-induced changes observed in hepatocytes are characteristic features of apoptosis. In contrast, the pretreatment of N-acetylcysteine (4 mM), a precursor of intracellular glutathione (GSH) and antioxidant, prevented PG (0.5 mM)-induced formation of soluble DNA fragments and loss of cellular GSH, ATP, and formation of blebbing. These results suggest that when the concentration of PG is decreased, the effects of PG on hepatocytes change from acute necrotic to apoptotic mode, and that the onset of DNA fragmentation is associated with GSH depletion. Received: 23 June 1997 / Accepted: 18 August 1997     

In silico binding of 4,4'‐bisphenols predicts in vitro estrogenic and antiandrogenic activity

Bisphenols, anthropogenic pollutants, leach from consumer products and have potential to be ingested and are excreted in waste. The endocrine disrupting effects of highly manufactured bisphenols (BPA, BPS, and BPF) are known, however the activities of others are not. Here, the estrogenic and androgenic activities of a series of 4,4'‐bisphenols that vary at the inter‐connecting bisphenol bridge were determined (BPA, BPB, BPBP, BPC2, BPE, BPF, BPS, and BPZ) and compared to in silico binding to estrogen receptor‐alpha and the androgen receptor. Bioassay results showed the order of estrogenicity (BPC2 (strongest) > BPBP > BPB > BPZ > BPE > BPF > BPA > BPS, r² = 0.995) and anti‐androgenicity (BPC2 (strongest) > BPE, BPB, BPA, BPF, and BPS, r² = 0.996) correlated to nuclear receptor binding affinities. Like testosterone and the anti‐androgen hydroxyflutamide, bisphenol fit in the ligand‐binding domain through hydrogen‐bonding at residues Thr877 and Asn705, but also interacted at either Cys784/Ser778 or Gln711 through the other phenol ring. This suggests the 4,4'‐bisphenols, like hydroxyflutamide, are androgen receptor antagonists. Hydrogen‐bond trends between ERα and the 4,4'‐bisphenols were limited to residue Glu353, which interacted with the –OH of one phenol and the –OH of the A ring of 17β‐estradiol; hydrogen‐bonding varied at the –OH of ring D of 17β‐estradiol and the second phenol –OH group. While both estrogen and androgen bioassays correlated to in silico results, conservation of hydrogen‐bonding residues in the androgen receptor provides a convincing picture of direct antagonist binding by 4,4'‐bisphenols.     

Age and Dose Dependency of the Pharmacokinetics and Metabolism of Bisphenol A in Neonatal Sprague-Dawley Rats Following Oral Administration

Previous studies demonstrated the rapid clearance of bisphenolA (BPA) from blood following oral administration to adult ratswith the principal metabolite being BPA-monoglucuronide (BPA-glucuronide).Since the ontogeny of glucuronyl transferases (GT) differs withage, the pharmacokinetics of BPA were studied in neonatal animals.¹⁴C-BPA was administered via gavage at 1 or 10 mg/kg body weightto rats at postnatal day (pnd) 4, pnd 7, pnd 21, or to 11 weekold adult rats (10 mg/kg dose only). Blood (neonates and adults)and selected tissues (neonates) were collected at 0.25, 0.75,1.5, 3, 6, 12, 18, and 24 h postdosing. BPA and BPA-glucuronidein the plasma were quantified by high-performance liquid chromatography;radioactivity in the plasma and tissues was quantified by liquidscintillation spectrometry. The data indicate that neonatalrats at all three ages metabolized BPA to BPA-glucuronide, althoughan age dependency in the number and concentration of plasmametabolites was observed, consistent with the ontogeny of GT.BPA-glucuronide and BPA concentrations in the plasma were greaterin neonates than in adults, except at 24 h postdosing, suggestingan immaturity in the development of hepatic excretory functionin neonatal rats. Nevertheless, the half-lives for the eliminationof BPA-glucuronide in plasma were more rapid in neonatal animalsthan in adults, likely due to reduced microflora ß-glucuronidaseactivity and an absence of enterohepatic recirculation. A dosedependency in the metabolism and pharmacokinetics of BPA administeredto neonates was also observed with nearly complete metabolismof BPA to BPA-glucuronide (94–100% of the plasma radioactivity)at a dose of 1 mg/kg. This was in contrast to finding up to13 different plasma metabolites observed at the 10 mg/kg dose.These data indicate that, from early in neonatal life throughpnd 21, there is sufficient GT activity in rats to efficientlymetabolize BPA to its nonestrogenic metabolite at low doses.     

Biotransformation and cytotoxicity of a brominated flame retardant, tetrabromobisphenol A, and its analogues in rat hepatocytes

The metabolism and cytotoxic effects of tetrabromobisphenol A (TBBPA), a phenolic flame retardant, and its analogues were studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria, respectively. The exposure of hepatocytes to TBBPA caused not only concentration (0.25-1.0 mM)- and time- (0-3 h) dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation. TBBPA at a weakly toxic level (0.25 mM) was metabolized to monoglucuronide and monosulfate conjugates: the amounts of glucuronide rather than sulfate conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In comparative effects based on cell viability, mitochondrial membrane potential and some toxic parameters, bisphenol A (BPA) was less toxic than TBBPA and tetrachlorobisphenol A (TCBPA), which are not significant differences in these parameters. In mitochondria isolated from rat liver, TBBPA and TCBPA caused an increase in the rate of State 4 oxygen consumption in the presence of succinate, indicating an uncoupling effect and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner (5-25 microM). Taken collectively, our results indicate that (i) mitochondria are target organelles for TBBPA, which elicits cytotoxicity through mitochondrial dysfunction related to oxidative phosphorylation at an early stage and subsequently lipid peroxidation at a later stage; and (ii) the toxicity of TBBPA and TCBPA is greater than that of BPA, suggesting the participation of halogen atoms such as bromine and chlorine in the toxicity.     

Male reproductive toxicity of four bisphenol antioxidants in mice and rats and their estrogenic effect

Male mice and rats were fed a diet containing four bisphenol antioxidants, 2,2′-methylenebis(4-ethyl-6-tert-butylphenol) (ME), 2,2′-methylenebis(4-methyl-6-tert-butylphenol) (MM), 4,4′-butylidenebis(3-methyl-6-tert-butylphenol) (BM), or 4,4′-thiobis(3-methyl-6-tert-butylphenol) (TM) at levels of 0.06–0.25% for 2 months. BM and TM decreased epididymal, seminal vesicular, prostate and preputial weights, and injured seminiferous tubules in mice in a dose-dependent fashion. BM and TM also reduced sex accessory organ weights and sperm production capacity in rats, but MM and ME were more toxic to rats than BM and TM. ME and MM did not bind ER_α up to 10⁻³ M, while BM and TM competitively bound ER_α against β-estradiol (E2). Fifty percent inhibitory concentrations (IC₅₀ s) of BM, TM, and bisphenol A (positive control) against E2-binding were 7.3×10⁻⁶ M, 1.8×10⁻⁵ M, and 1.4×10⁻⁵ M, respectively. When ovariectomized (OVX) mice were sc administered TM at doses of 60 and 300 mg/kg/day for 4 days, or when OVX mice were fed BM in the diet at a level of 0.25% for 2 months, uterine weight was significantly increased. These results suggest that BM and TM are weakly toxic, possibly through an estrogenic mechanism to male reproductive organs in mice as well as rats, while MM and ME may be the direct testicular toxins in rats but not mice. A part of this study was presented at the 74th annual meeting of the Japanese Society for Hygiene held from 24–27 March 2004 in Tokyo, Japan.     

Characterization of CYP1A in hepatocytes of cynomolgus monkeys (Macaca fascicularis) and induction by different substituted polychlorinated biphenyls (PCBs)

Cynomolgus monkeys (Macaca fascicularis) have been used previously as a model to study effects on cytochrome P450 (CYP) regulation. Until now it has not been elucidated which CYP1A proteins are present in this primate species. The aim of this study was to characterize CYP1A in untreated hepatocytes of cynomolgus monkey using two specific CYP1A inhibitors (α -naphthoflavone and furafylline). The effect of different substituted polychlorinated biphenyls (PCBs) on CYP1A regulation was also studied in these hepatocytes. Small quantities of CYP1A2 have been identified in untreated hepatocytes. Northern blots showed the presence of a CYP1A mRNA in untreated hepatocytes, when hybridizations where performed with human CYP1A2 cDNA. Inhibitions with furafylline and α -naphthoflavone also suggested the presence of CYP1A2 properties. After induction with different PCBs, (probably) CYP1A1 mRNA and enzyme activity were induced in cynomolgus monkey hepatocytes. As expected, 2,3′,4,4′,5-PeCB (PCB no. 118), a mono-ortho substituted congener, was a potent CYP1A inducer but 2,2′,3,4,4′,5′,5′-HpCB (PCB no. 180), a di-ortho and 2,2′,3,4′,5,5′,6-HpCB (PCB no. 187), a tri-ortho substituted PCB, could induce CYP1A mRNA and enzyme activity in cynomolgus monkey hepatocytes as well. Received: 20 April 1998 / Accepted: 1 July 1998     

Chlorpropham induces mitochondrial dysfunction in rat hepatocytes

The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) and its metabolites were studied in freshly isolated rat hepatocytes and isolated rat hepatic mitochondria, respectively. The exposure of hepatocytes to CIPC caused a concentration (0.25–1.0 mM)- and time (0–3 h)-dependent cell death accompanied by loss of cellular ATP and adenine nucleotides. CIPC at a weakly toxic level (0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to a minor metabolite 3-chloroaniline (3CA). The addition of SKF-525A (50 μM), an inhibitor of microsomal monooxygenase, enhanced the CIPC (0.5 mM)-induced cytotoxicity accompanied by loss of ATP and 4OH-CIPC and inhibited the decrease in the concentration of the parent compound. CIPC led to a strong decrease in cellular ATP content compared to its metabolites, 4OH-CIPC and 3CA. On the other hand, the exposure of isolated hepatic mitochondria to CIPC reduced State 3 respiration with a FAD-linked substrate (succinate plus rotenone) and/or with a NAD⁺-linked substrate (pyruvate plus malate), whereas State 3 respiration with ascorbate plus tetramethyl-p-phenylendiamine (cytochrome oxidase-linked respiration) was not affected markedly by CIPC. Further, the addition of CIPC caused an increase in the rate of State 4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner, respectively. In contrast, the addition of neither 4OH-CIPC nor 3CA markedly affected the rate of states 3 and/or 4 oxygen consumption. These results indicate that CIPC-induced cytotoxicity is mediated by the parent compound rather than by its metabolites such as 4OH-CIPC and 3CA, and that the toxicity is associated with a rapid depletion of ATP via impairment of mitochondrial function related to oxidative phosphorylation.     

Metabolism of bisphenol A in isolated rat hepatocytes and oestrogenic activity of a hydroxylated metabolite in MCF-7 human breast cancer cells

1. The metabolites of bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane) in freshly isolated rat hepatocytes and the oestrogenic activities of BPA and its metabolites, particularly 3-hydroxybisphenol A (3-OH-BPA), in MCF-7 cells and competitive binding assays have been studied, respectively. 2. During a 2-h incubation, almost all of the BPA (0.25 mM) added to the hepatocyte suspensions was rapidly converted to a major conjugate, monoglucuronide (approximately 75% of total metabolites), and two minor conjugates, which were tentatively identified as monosulphates of BPA and a hydroxylated intermediate, 3-OH-BPA, as determined by mass spectroscopy coupled with HPLC or GC/MS. On the other hand, free 3-OH-BPA was identified as a trace metabolite, whose level was approximately 1 or 2 microM at 1 h in hepatocyte suspensions treated with 0.25 or 0.5 mM BPA, respectively. 3. In another experiment, 3-OH-BPA as well as BPA displaced competitively 17beta-oestradiol bound to the recombinant human oestrogen receptor alpha in a concentration dependent-manner: IC50 of diethylstilbestrol, BPA and 3-OH-BPA were approximately 2.5 x 10(-8), 10(-5) and 5 x 10(-5) M, respectively. Further, BPA and 3-OH-BPA at intermediate concentrations (10(-7) - 10(-6) M) caused proliferation of MCF-7 human breast cancer cells, whereas the effect of BPA was more potent than that of 3-OH-BPA. At higher concentrations, both BPA (> 10(-4)) and 3-OH-BPA (> 10(-5) M) were cytotoxic. 4. Based on the proliferative potency in MCF-7 cells and the IC50 for the competitive binding, the oestrogenic activity of 3-OH-BPA was less than that of BPA. These results indicate that BPA itself rather than its metabolite acts as a xeno-oestrogen and that 3-OH-BPA is cytotoxic, possibly acting via reactive semiquinone and/or quinone metabolites, rather than a xeno-oestrogenic mechanism, in MCF-7 cells.     

Carnosine prevents glyceraldehyde 3-phosphate-mediated inhibition of aspartate aminotransferase

Post-mitotic tissues, such as the heart, exhibit high concentrations (20 mM) of carnosine (β-alanyl-l-histidine). Carnosine may have aldehyde scavenging properties. We tested this hypothesis by examining its protective effects against inhibition of enzyme activity by glyceraldehyde 3-phosphate (Glyc3P). Glyc3P is a potentially toxic triose; Glyc3P inhibits the cardiac aspartate aminotransferase (cAAT) by non-enzymatic glycosylation (or glycation) of the protein. cAAT requires pyridoxal 5-phosphate (PyP) for catalysis. We observed that carnosine (20 mM) completely prevents the inhibition of cAAT activity by Glyc3P (5 mM) after brief incubation (30 min at 37 °C). After a prolonged incubation (3.25 h) of cAAT with Glyc3P (0.5 mM) at 37 °C, the protection by carnosine (20 mM) persisted but PyP availability was affected. In the absence of PyP from the assay medium, cAAT activities (plus Glyc3P) were 95 ± 18.2 μmol/min per mg protein (mean ± SD), minus carnosine and 100 ± 2.4, plus carnosine; control activity was 172 ± 3.9. When PyP (1.0 μM) was included in the assay medium, cAAT activities (plus Glyc3P) were 93 ± 14.8, minus carnosine and 151 ± 16.8, plus carnosine, P < 0.001; control activity was 180 ± 17.7. These data, which showed carnosine moderating the effects of both Glyc3P and PyP, suggest that carnosine may be an endogenous aldehyde scavenger. Received: 30 March 1999 / Accepted: 3 May 1999     

Cytotoxic and xenoestrogenic effects via biotransformation of trans-anethole on isolated rat hepatocytes and cultured MCF-7 human breast cancer cells

The metabolism and action of trans-anethole (anethole) and the estrogen-like activity of the compound and its metabolites were studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with anethole (0.25-2.0mM) caused a concentration- and time-dependent cell death accompanied by losses of cellular ATP and adenine nucleotide pools. Anethole at a weakly toxic level (0.5mM) was metabolized to 4-methoxycinnamic acid (4MCA), 4-hydroxy-1-propenylbenzene (4OHPB), and the monosulfate conjugate of 4OHPB; the levels of 4OHPB sulfate and 4MCA reached approximately 20 and 200 microM within 2 hr, respectively, whereas that of free unconjugated 4OHPB was less than approximately 0.5 microM. At a moderately toxic concentration (1.0mM), unconjugated 4OHPB reached approximately 10 microM, followed by abrupt loss of 3'-phosphoadenosine 5'-phosphosulphate (PAPS). Based on cell viability and adenine nucleotide levels, 4OHPB was more toxic than anethole and 4MCA. The addition of 2,6-dichloro-4-nitrophenol (50 microM), an inhibitor of sulfotransferase, enhanced the anethole-induced cytotoxicity associated with losses of ATP, PAPS, and 4OHPB sulfate, and symmetrically increased the unconjugated 4OHPB concentration. 4OHPB as well as diethylstilbestrol (DES) and bisphenol A (BPA), which are known xenoestrogenic compounds, competitively displaced 17beta-estradiol bound to the estrogen receptor alpha in a concentration-dependent manner; IC(50) values of these compounds were approximately 1 x 10(-5), 1 x 10(-8) and 5 x 10(-5)M, respectively. 4OHPB also caused a concentration (10(-8) to 10(-6)M)-dependent proliferation of MCF-7 cells, whereas neither anethole nor 4MCA (10(-9) to 10(-5)M) affected cell proliferation. However, at higher concentrations (>10(-4)M), 4OHPB rather than anethole and 4MCA was cytotoxic. These results suggest that the biotransformation of anethole induces a cytotoxic effect at higher concentrations in rat hepatocytes and an estrogenic effect at lower concentrations in MCF-7 cells based on the concentrations of the hydroxylated intermediate, 4OHPB.     

Enhanced OECD TG 407 in detection of endocrine-mediated effects of 4,4′-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol

The purpose of this study was to investigate whether the estrogenic effects were detected in the enhanced TG 407 if the estrogenic property was not so strong in the uterotrophic assay. The estrogenic property of 4,4′-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol in the uterotrophic assay was slightly stronger than that of genistein or nonylphenol, but weaker than that of ethinyl estradiol. We performed a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) on 4,4′-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol based on the OECD draft protocol. The test chemical, administered orally at doses of 0, 10, 50, and 250 mg/kg per day for at least 28 days, caused such estrogenic effects as abnormal estrous cycle, increased ovarian follicles, increased uterine epithelial height, and vaginal mucification in the 50 and/or 250 mg/kg groups. Moreover, follicular epithelial cell hyperplasia of the thyroid was detected in all male rats given the test chemical and in female rats in the 250 mg/kg group. It was concluded that the estrogenic effects were detected in growing rats given 4,4′-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, and thyroid dysfunction was also observed as the endocrine-mediated effects.     

Biotransformation of chlorpropham (CIPC) in isolated rat hepatocytes and xenoestrogenic activity of CIPC and its metabolites by in vitro assays

1: The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) were studied in freshly isolated rat hepatocytes, and the oestrogen-like activity of CIPC and its metabolites was assessed by in vitro assays. The exposure of hepatocyte suspensions to CIPC caused concentration- (0.25-1.0 mM) and time- (0-3 h) dependent cell death, which was assessed by Trypan blue exclusion, accompanied by losses of cellular adenosine triphosphate and adenine nucleotide pools, and formation of cell bleb. 2: CIPC at a weakly toxic level (0.25 or 0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to the minor metabolites 3-chloroaniline (3CA) and 3-chloroacetanilide. CIPC (0.25 mM) added to hepatocyte suspensions was distributed equally between hepatocytes and the extracellular medium during the incubation. The glucuronide rather than the sulfate conjugate of 4OH-CIPC predominantly increased in the medium with time, while the amount of unconjugated free 4OH-CIPC in the extracellular medium increased by approximately threefold compared with the amount in the cell fraction after 0.5 h and then decreased rapidly accompanied by increases in the conjugates. This indicates that unconjugated free 4OH-CIPC produced in hepatocytes was temporarily excreted in the extracellular medium and subsequently converted to the conjugates via re-influx into hepatocytes. 3: Diethylstilbestrol (DES), bisphenol A (BPA) and 4-hydroxybenzoic acid butyl ester (butylparaben), which are known xenoestrogenic compounds, competitively displaced 17beta-oestradiol bound to the oestrogen receptor-alpha (ERalpha) in a concentration-dependent manner; IC50 values of DES, BPA, butylparaben and its derivative 3-chloro-4-hydroxybenzoic acid butyl ester (3-chloro-butylparaben) were approximately 10(-8), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. In contrast, neither CIPC nor 4OH-CIPC impaired the binding of 17beta-oestradiol to ERalpha at concentrations ranging from 10(-9) to 10(-4) M, whereas at concentrations of >5 x 10(-4) M, the binding affinity of 4OH-CIPC was greater than that of CIPC. In a proliferation assay of MCF-7 cells, CIPC, 4OH-CIPC and 3CA did not increase cell numbers at concentrations ranging from 10(-9) to 10(-5) M, but these compounds at a concentration of 10(-4) M induced a considerable decrease in cell numbers relative to the control. The results suggest that even if CIPC is metabolized to 4OH-CIPC by hepatocytes, the chlorine adjacent to the 4-hydroxy group added to the intermediate as well as 3-chloro-butylparaben obstructs the appearance of oestrogen-like effects via an interaction between the intermediate and the ER.     

Effects of hexavalent chromium on trout mitochondria

Trout liver mitochondria were incubated in the presence of micromolar concentration of potassium dichromate (Cr(VI)) under several experimental conditions. Cr(VI) strongly inhibited both state 3 and state 4 of respiration supplemented by NAD-linked substrates; it also slightly affected the respiration of FAD-linked substrates. Evidence is provided that the respiratory inhibition induced by dichromate is partially coupled to the Cr(VI) reduction mechanism occurring in mitochondria.     

Elucidation of mitochondrial effects by tetrahydroaminoacridine (tacrine) in rat, dog, monkey and human hepatic parenchymal cells

Tetrahydroaminoacridine (tacrine) causes morphological and functional changes in the endoplasmic reticulum, ribosomes, and mitochondria in the liver of humans and animals. In order to investigate species differences as well as to understand the morphological changes, we examined the effects of tacrine on respiration and electron transport in mitochondria isolated from rat, dog, monkey, and human liver. Tacrine produced significantly decreased respiratory control ratios (RCR) in all species at concentrations ranging from 5 to 25 μg/ml. Human mitochondria were more sensitive to tacrine effects with RCR decreased 24% at 5 μg/ml while other species were unaffected at this concentration. The tacrine effects were characterized by increased hepatic mitochondrial State 4 respiration in rats and decreased State 3 respiration in humans. Mitochondria from aged rats were more sensitive to the effects of tacrine than mitochondria from young animals, with significantly decreased RCR at 10 μg/ml in aged rats while mitochondria from young rats were unaffected at this concentration. Concomitant with the respiratory changes, mitochondrial DNA synthesis was impaired. Since tacrine undergoes extensive biotransformation, we also explored the possibility that metabolites could exert detrimental effects. The ranking order of potency for decreasing RCR caused by monohydroxylated metabolites was: tacrine >4-OH and 7-OH >2-OH, 1-OH, and velnacrine with the latter group of metabolites having no effect on mitochondrial respiration at concentrations up to 50 μg/ml. In vivo administration of 20 mg/kg tacrine to rats for up to 20 days caused a paradoxical increase in RCR and P/O on Day 1 and decreased RCR on Days 9 and 20, the later findings being consistent with in vitro data. From these data we propose that tacrine does not necessarily have to be metabolized to exert effects on mitochondria at different sites in the electron transport chain that differ among species. These effects are exacerbated in mitochondria from older animals and humans appear to be more sensitive than the laboratory animals studied. Received: 23 September 1997 / Accepted: 17 November 1997     

Metabolism and pharmacokinetics of bisphenol A (BPA) and the embryo-fetal distribution of BPA and BPA-monoglucuronide in CD Sprague-Dawley rats at three gestational stages.

The pharmacokinetics of bisphenol A (BPA), including the quantification of the major BPA metabolite BPA-monoglucuronide conjugate (BPA-glucuronide) was studied in Sprague-Dawley rats at different stages of gestation. 14C-BPA was administered orally at 10 mg BPA/kg body weight (0.2 mCi/rat) to nongravid rats and to other groups on gestation days (GD) 6, 14, and 17. GD 0 was when the vaginal smear was sperm positive or a copulatory plug was observed. Radioactivity derived from 14C-BPA was quantified in the maternal blood, selected tissues, and the embryo or fetus. BPA and BPA-glucuronide were quantified in maternal plasma and excreta. Additional rats were dosed orally at 10 mg 14C-BPA/kg (0.2 mCi/rat or 0.5 mCi/rat) on GD 11, 13, and 16 to further study the distribution of BPA and BPA-glucuronide to the embryo/fetal tissue. The tissue distribution, metabolism, or the rates or routes of excretion of BPA, or the plasma concentration-time profiles of BPA-glucuronide did not appear to be altered at any stage of gestation as compared to nonpregnant rats. In the GD 11 group, neither BPA nor BPA-glucuronide was detected in the yolk sacs or embryos, except for trace concentrations of BPA-glucuronide in the yolk sacs at 15 min postdosing. In the GD 13 group, both BPA and BPA-glucuronide were detected in the yolk sacs of the conceptus but not in the embryos/fetuses, except for BPA at 15 min. For the animals dosed with 0.2 mCi/rat on GD 16, both analytes were detected in the placentae at 15 min and 12 h, but not at 96 h. Traces of both analytes were detected in fetal tissue in two of five specimens at 15 min only. In rats dosed on GD 16 with 0.5 mCi/rat, the BPA-glucuronide and BPA concentrations in maternal plasma at 15 min were 1.7 and 0.06 mug equivalents (eq)/g plasma, respectively. At the same time postdosing in these animals, the placental BPA-glucuronide concentrations were lower (0.34 mug eq BPA [as glucuronide]/g), and the BPA concentrations were about equivalent (0.095 mug/g). Fetal BPA-glucuronide and BPA concentrations were markedly lower, 0.013 and 0.018 mug eq/g, respectively. Therefore, no selective affinity of either yolk sac/placenta or embryo/fetus for BPA or BPA metabolites relative to maternal plasma or tissues was observed in this study.     

Congener specific effects by polychlorinated biphenyls on catecholamine content and release in chromaffin cells

The effects of the non-planar polychlorinated biphenyl (PCB) congener 2,2′,4,4′-tetrachlorobiphenyl (2,4-TCB) and of the coplanar PCB congener 3,3′,4,4′-tetrachlorobiphenyl (3,4-TCB) were investigated on the catecholamine content and release from bovine adrenal chromaffin cells in culture. Each congener was tested at three concentrations (20, 50 and 100 μM) and two exposure periods (24 h and 5 days). Catecholamine release induced by K⁺-stimulation as well as catecholamine content of Triton X-100 treated cell cultures were examined using high-performance liquid chromatography (HPLC). 2,4-TCB showed dose- and time-dependent effects. 2,4-TCB at 100 μM reduced the K⁺-stimulated catecholamine release after 24 h of exposure. After 5 days of exposure, 2,4 TCB at 50 and 100 μM drastically reduced the K⁺-stimulated catecholamine release. 3,4-TCB even at a concentration of 100 μM over exposure of either 24 h or 5 days had no effects on the K⁺-stimulated secretion. When chromaffin cells, exposed to 2,4-TCB, were lysed with 0.5% Triton X-100, a dose- and time-dependent reduction of the catecholamine content appeared. The 3,4-TCB did not reduce the catecholamine content. Conversely there seemed to be a trend towards an increase in catecholamine content. Spontaneous release of catecholamines was strongly increased by the non-planar 2,4 TCB, while the coplanar 3,4 TCB showed no effects on this parameter. Furthermore, the effects of 2,4 TCB appeared to be reversible after replacing the highest concentration (100 μM) of the TCB-solution with culture-medium at the end of the 24-h exposure. Thus, K⁺-stimulated catecholamine release and the catecholamine content of bovine adrenal chromaffin cells was effectively reduced by the non-planar PCB congener whereas spontaneous catecholamine release was strongly increased. The coplanar PCB congener was ineffective at the same conditions. Received: 7 January 1997 / Accepted: 11 February 1997     

Effects of cocaine and its oxidative metabolites on mitochondrial respiration and generation of reactive oxygen species

Cocaine is capable of producing severe hepatocellular necrosis in laboratory animals and humans. The mechanism of cocaine hepatotoxicity is not well understood, but appears to result from the actions of one or more N-oxidative metabolites of cocaine. Mitochondria have been proposed as critical cellular targets for cocaine toxicity, and previous studies have found depressed mitochondrial respiration and increased mitochondrial generation of reactive oxygen species (ROS) in animals treated with cocaine. To examine the potential role of cocaine N-oxidative metabolites in these effects, mitochondrial respiration and ROS generation were examined in isolated mouse mitochondria treated with cocaine and its N-oxidative metabolites-norcocaine, N-hydroxynorcocaine, and norcocaine nitroxide. Cocaine, in concentrations of 0.25 or 0.5 mM, had no effect on state 3 respiration, state 4 respiration, respiratory control ratio (RCR), or ADP/O ratio. Norcocaine (0.5 mM) inhibited state 3 respiration, and N-hydroxynorcocaine (0.5 mM) inhibited both state 3 and state 4 respiration. Norcocaine nitroxide had the greatest effect on mitochondrial respiration; the lower concentration (0.25 mM) completely inhibited both state 3 and state 4 respiration. Preincubation of mitochondria with cocaine or metabolites increased the inhibitory effect of norcocaine and N-hydroxynorcocaine, but not cocaine. Cocaine, norcocaine, and N-hydroxynorcocaine (0.1 mM) had no effect on ROS generation during state 3 respiration, and cocaine and norcocaine decreased ROS generation under state 4 conditions. Norcocaine nitroxide interfered with the fluorescence ROS assay and could not be assessed. The results suggest that the effects of cocaine on mitochondrial respiration are due to its N-oxidative metabolites. Inhibition of mitochondrial respiration by the N-oxidative metabolites of cocaine may be the underlying cause for observed ATP depletion and subsequent cell death.     

Bisphenols B,E, F,and S and 4‐cumylphenol induce lipid accumulation in mouse adipocytes similarly to bisphenol A

Bisphenol A (BPA) has been widely reported to exert endocrine disrupting effects, including the induction of adipogenesis in cultured preadipocytes and intact animals. Because of the potential harm to human health, BPA is being substituted by structurally related bisphenols. Whether or not such BPA analogues are safe substitutes, however, remains largely unknown. Here, we investigated the potential of bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF), bisphenol S (BPS), and 4‐cumylphenol (4‐CP) to affect lipid and hormone levels in 3 T3‐L1 cells. We found that BPB, BPE, BPF, BPS, and 4‐CP all affected lipid accumulation and leptin levels to the same extent and potencies as BPA. Based on these and other results, we conclude that these BPA analogues and 4‐CP most likely will elicit similar effects on adipocytes as BPA. Using them to substitute BPA in products should be done with caution.     

Induction of CYP2A5 by pyrazole and its derivatives in mouse primary hepatocytes

Mouse liver CYP2A5 is induced by several structurally unrelated compounds. In intact mouse liver, pyrazole (PYR) and 4-hydroxypyrazole (4-OH) induce selectively the expression of CYP2A5 while expression of other CYPs is decreased. In this study we exposed mouse primary hepatocytes to PYR, 4-OH, 4-methylpyrazole (4Me; 0.1–20 mM) and 4-iodopyrazole (4-I; 0.1–5.0 mM). PYR and its derivatives increased coumarin 7-hydroxylase activity, with 4-1 and 4-OH being the strongest inducers, by 114-fold and 41-fold, respectively. However, only 4-1 treatment increased markedly the CYP2A5 protein content. CYP2B9/10-mediated pentoxyresorufin O-deethylase activity (PROD) was decreased by 80% by 4-Me and 4-1, and by 50% by 4-OH while PYR had no marked effect. PYR and 4-Me␣increased 2- to 3-fold the CYPA1/2-mediated ethoxyresorufin O-deethylase activity (EROD) while 4-OH and 4-1 had no marked effect on this enzyme. The time of exposure markedly affected the inducibility of 4-OH such that induction was 7-fold stronger when it was added to the incubation medium 24 h after the isolation of hepatocytes compared to exposure 3 h after their isolation. Cimetidine prevented the induction of coumarin 7-hydroxylase activity by PYR and 4-OH by 46 and 74%, respectively indicating that their effects on the expression of CYP2A5 are, at least partly, mediated via their metabolites. The data demonstrate that the regulation of CYP2A5 is different from other monooxygenases and that the effects of pyrazole and its derivatives are different in vivo and in vitro. Also, the timing of exposure markedly affects the inducibility of 4-OH in hepatocytes. Received: 22 September 1997 / Accepted: 13 January 1998