Toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic cytochrome P450 in F344 rats

The subacute toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic microsomal cytochrome P450 (P450) were investigated in male and female F344 rats. Rats were given 0.25, 0.5, and 1.0% terbutol for 28 days. Liver weights of male and female rats increased at all dose levels. The compound did not affect activity or amount of serum biochemical markers related to hepatic damage. The concentrations of terbutol in rat serum were less than 0.1 microM, and its major metabolites in serum were 2,6-di-tert-butyl-4-carboxyphenyl N-methyl-carbamate and 2,6-di-tert-butyl-4-carboxyphenol. In male rats, P450 and cytochrome b5 (b5) contents, and NADPH cytochrome c reductase (fp2) activity in liver microsomes were increased about 2-fold by 1% terbutol administration for 7 to 28 days. Among the P450-dependent monooxygenase activities in liver microsomes, 7-benzyloxyresorufin-O-debenzylase (BROD) activity was greatly increased by 100-fold, and 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), and aminopyrine-N-demethylase (APND) activities were elevated 2- to 3-fold. 7-Methoxyresorufin-O-deethylase (MROD), erythromycin-N-demethylase (EMND), estradiol 2-hydroxylase (ED2H), chlorzoxazone 6-hydroxylase (CZ6H), and lauric acid omega-hydroxylase (LAOH) activities were unchanged. For the activities of testosterone hydroxylation, testosterone 16beta-hydroxylase (T16BH) activity was markedly increased by 30-fold, and testosterone 6beta-hydroxylase (T6BH) and testosterone 7alpha-hydroxylase (T7AH) activities were slightly elevated. Testosterone 2alpha-hydroxylase (T2AH) activity was not affected. Terbutol 4-methylhydroxylase (T4MH) activity was increased 9-fold by 1% terbutol. In an immunoinhibition study, T4MH activity in liver microsomes from 1% terbutol-treated rats was decreased about 50% by polyclonal anti-rat CYP2B1, whereas polyclonal anti-rat CYP2A1 and CYP2C11 did not affected the activity. These results indicate that terbutol increased CYP2B subfamily in rat liver microsomes, and that the compound did not cause serious hepatic damage.     

Percutaneous absorption of 2,6-di-tert-butyl-4-nitrophenol (DBNP) in isolated perfused porcine skin.

DBNP (2,6-di-tert-butyl-4-nitrophenol) has been reported as a potential contaminant in submarines. This yellow substance forms when lubrication oil mist containing the antioxidant additive 2,6-di-tert-butylphenol passes through an electrostatic precipitator and is nitrated. Percutaneous absorption of 14C-DBNP was assessed in the isolated perfused porcine skin flap (IPPSF). Four treatments were studied (n=4 flaps/treatment): 40.0 microgram/cm(2) in 100% ethanol; 40.0 microgram/cm(2) in 85% ethanol/15% H(2)O; 4.0 microgram/cm(2) in 100% ethanol; and 4.0 microgram/cm(2) in 85% ethanol/15% water. DBNP absorption was minimal across all treatment groups, with the highest absorption detected being only 1.08% applied dose in an aqueous ethanol group. The highest mass of 14C-DBNP absorbed was only 0.5 microgram. The majority of the applied dose remained on the surface of the skin. This suggests that there is minimal dermal exposure of DBNP when exposed topically to skin.     

Antiinflammatory 2,6-di-tert-butyl-4-(2-arylethenyl)phenols

A series of 2,6-di-tert-butyl-4-(2-arylethenyl)phenols was prepared and examined for their ability to inhibit cyclooxygenase and 5-lipoxygenase in vitro and developing adjuvant arthritis in vivo in the rat. Structure-activity relationships are discussed. Among the best compounds is (E)-2,6-di-tert-butyl-4-[2-(3-pyridinyl)ethenyl]phenol (7d). It has an IC50 of 0.67 microM for cyclooxygenase and 2.7 microM for 5-lipoxygenase and an ED50 of 2.1 mg/kg in developing adjuvant arthritis. Additional in vivo data are reported for 7d.     

Cytotoxic effects of thioxanthone derivatives as photoinitiators on isolated rat hepatocytes

Thioxanthone and its analogues, 2- or 4-isopropylthioxanthone, 2-chlorothioxanthone , 2,4-diethylthioxanthone (DETX) and xanthone, are used as photoinitiators of ultraviolet (UV) light-initiated curable inks. As these photoinitiators were found in numerous food/beverage products packaged in cartons printed with UV-cured inks, the cytotoxic effects and mechanisms of these compounds were studied in freshly isolated rat hepatocytes. The toxicity of DETX was greater than that of other compounds. DETX elicited not only concentration (0–2.0 mm )- and time (0–3 hours)-dependent cell death accompanied by the depletion of cellular adenosine triphosphate (ATP), and reduced glutathione (GSH) and protein thiol levels, but also the accumulation of GSH disulfide and malondialdehyde. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or N-acetyl-l -cysteine (NAC) at a concentration of 5.0 mm ameliorated DETX (1 mm )-induced cytotoxicity. Further, the exposure of hepatocytes to DETX resulted in the induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential, both of which were partially prevented by the addition of NAC. These results indicate that: (1) DETX-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were, at least in part, ameliorated by the addition of fructose; and (3) GSH loss and/or ROS formation was prevented by NAC. Taken collectively, these results suggest that the onset of toxic effects caused by DETX may be partially attributable to cellular energy stress as well as oxidative stress.     

Cytotoxic effects of phenyl-hydroquinone and some hydroquinones on isolated rat hepatocytes.

The cytotoxic effects of phenyl-hydroquinone (PHQ) and some other hydroquinones on freshly isolated rat hepatocytes were investigated. Addition of PHQ (0.5 or 0.75 mM) to the hepatocytes elicited dose-dependent cell death accompanied by losses of intracellular glutathione (GSH), protein thiols and ATP. These effects were related to both PHQ loss and phenyl-benzoquinone (PBQ) formation in the cell suspension. The cytotoxicity of PHQ was prevented by sulphydryl compounds such as cysteine and GSH. In Krebs-Henseleit buffer without cells, loss of PHQ (0.5 mM; initial concentration) and formation of PBQ, monitored by spectral measurements, were inhibited by addition of 50 microM GSH. Further, the oxygen consumption owing to autoxidation of PHQ (0.5 mM) in Krebs-Henseleit buffer without cells was depressed by addition of 50 microM GSH. Among all the hydroquinones tested (at 0.5 mM), tert-butyl-hydroquinone and PHQ were most toxic, followed by hydroquinone and 2,5-di(tert-butyl)-1,4-benzohydroquinone. However, accumulation of cellular malondialdehyde was not affected by these hydroquinones. The toxicity was related to the rate of oxygen consumption by each hydroquinone in the buffer. These results suggest that hydroquinone-induced cytotoxicity is dependent on the rate of oxidation of these compounds as well as the loss of protein thiols.     

Uptake of perfluorooctanoate in freshly isolated hepatocytes from male and female rats

Liver is a primary target organ for perfluorooctanoate (PFO, the deprotonated form of perfluorooctanoic acid, PFOA) distribution in both male and female rats. We studied the uptake of PFO in freshly isolated hepatocytes from male and female rats. We identified a non-saturable cell partitioning process for PFO using on-ice incubations. At 37 degrees C, hepatic uptake of PFO was composed of the non-saturable partition as well as a saturable, active uptake process. The K(m) and V(max) values for the active uptake process were 88.0+/-9.1muM and 5.61+/-0.88nmol/(min10(6)cells), respectively, for male rat hepatocytes, and 76.1+/-12.0muM and 3.59+/-0.29nmol/(min10(6)cells), respectively, for female rat hepatocytes. The values of PFO clearance by active uptake were 64.8+/-15.7 and 47.6+/-4.7muL/(min10(6)cells) for male and female rat hepatocytes, respectively. The active uptake of PFO in rat hepatocytes was inhibited by sulfobromophthalein, a known substrate of organic anion transporting polypeptides, with apparent inhibition constants of 85.9+/-25.1 and 29.3+/-19.2muM in male and female rat hepatocytes, respectively. When serum albumin was added to the incubations, PFO hepatic uptake rates were reduced, but were proportional to the unbound fractions of PFO.     

Cytotoxic effects of hydroxylated fullerenes on isolated rat hepatocytes via mitochondrial dysfunction

The cytotoxic effects of hydroxylated fullerenes, also termed fullerenols or fullerols [C₆₀(OH) _n ], which are known nanomaterials and water-soluble fullerene derivatives, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C₆₀(OH)₂₄ caused not only concentration (0–0.25 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs, loss of cellular ATP, reduced glutathione (GSH), and protein thiol levels, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. Of the other analogues examined, the cytotoxic effects of C₆₀(OH)₁₂ and fullerene C₆₀ at a concentration of 0.125 mM were less than those of C₆₀(OH)₂₄. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes incubated with C₆₀(OH)₂₄ were greater than those with C₆₀(OH)₁₂ and fullerene C₆₀. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration were more markedly decreased by C₆₀(OH)₂₄ and C₆₀(OH)₁₂ compared with C₆₀. In addition, C₆₀(OH)₂₄ and C₆₀(OH)₁₂ resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of C₆₀(OH)₁₂ were less than those of C₆₀(OH)₂₄. Taken collectively, these results indicate that (a) mitochondria are target organelles for fullerenols, which elicit cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis in the early stage and subsequently oxidation of GSH and protein thiols, and lipid peroxidation through oxidative stress at a later stage; and (b) the toxic effects of fullerenols may depend on the number of hydroxyl groups participating in fullerene in rat hepatocytes.     

Lack of effect of insulin in hepatocytes isolated from streptozotocin-diabetic male rats

Diabetes mellitus is known to affect drug and steroid metabolism in the rat liver. Recently we have demonstrated that in-vitro insulin addition to hepatocytes obtained from normal male rats showed a significant dose-related increase in androstenedione metabolism. We have extended our study this time by using 3- and 21-days streptozotocin (STZ) diabetic and insulin-treated STZ-diabetic male rats. Hepatocytes from 3- and 21-days STZ-diabetic rats were resistant to the effect of insulin while insulin-treated diabetic rats indicated partial restoration of insulin effect. Since insulin resistance is a characteristic feature of type II diabetes mellitus, we would like to suggest that STZ-diabetic rats may be a model for type II diabetes mellitus.     

2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT quinone methide): an active metabolite of BHT causing haemorrhages in rats

Male Sprague-Dawley rats and male ICR mice, species respectively susceptible and resistant to the haemorrhagic effect of butylated hydroxytoluene (BHT), were administered BHT quinone methide (2,6-di-tert-butyl4-methylene-2,5-cyclohexadienone) orally; 24 or 48 h later the plasma concentrations of blood coagulation factors II (prothrombin), VII, IX and X were determined. BHT quinone methide caused a decrease in factors II, VII, IX and X in a dose-dependent manner after 48 h, while a similar dose of BHT did not. Haemorrhages in epididymis or thymus were found in BHT quinone methide-treated rats. These findings may support the belief that BHT quinone methide is an active metabolite which disturbs the vitamin K redox cycle in BHT-induced haemorrhage.     

The toxicity of disulphides to isolated hepatocytes and mitochondria

The disulfide metabolites of thiono-sulfur drugs were found to be about 50 to 100 times more toxic to isolated rat hepatocytes than the corresponding parent drugs. The order of decreasing cytotoxicity for the disulfide metabolites was disulfiram greater than propylthiouracil disulfide greater than formamidine disulfide greater than phenylthiourea disulfide greater than thiobenzamide disulfide greater than cystamine. Depletion of intracellular GSH levels preceded cytotoxicity. GSH could be restored and cytotoxicity averted by adding the thiol reducing dithiothreitol. Depletion of GSH with diethylmaleate potentiated the toxicity of disulfides 3 to 4-fold confirming the protective role of GSH in disulfide toxicity. The toxicity of disulfiram was increased 4-fold in cells pretreated with ATP (0.8 mM) to effect a transient increase in cytosolic Ca2+ suggesting an impairment of Ca2+ homeostasis by the toxicant. Disulfiram (200 microM) rapidly depleted hepatocyte ATP levels within 15 minutes which suggests that ATP production is inhibited. The disulfide effectiveness at causing mitochondrial Ca2+ release was similar to their effectiveness at inducing hepatocyte cytotoxicity. These results suggest that hepatocyte toxicity is the result of oxidative inactivation of membrane protein thiols that regulate intracellular Ca2+ homeostasis.     

Subchronic oral toxicity of a combination of insecticide (HCH) and herbicide (ISP) in male rats

Rats were treated orally with technical hexachlorocyclohexane (HCH, 12.5, 25 and 50 mg kg-1 day(-1)) and technical isoproturon (ISP 22.5. 45 and 90 mg kg-1 day(-1)) daily for a period of 90 days alone and in combination. Treatment with HCH alone showed mild to severe toxicity and death. Significant changes occurred in liver weight, clinical enzyme profiles, haematological parameters and pathomorphological changes. Treatment with ISP alone did not produce such changes. The combination of HCH and ISP produced changes not suggestive of synergism.     

The interaction of oral hypoglycaemic drugs with insulin on steroid metabolism in hepatocytes isolated from control and diabetic male rats

The effects of the oral hypoglycaemic drugs, phenformin and tolbutamide, and insulin, alone and in combination, on steroid metabolism in hepatocytes isolated from control and streptozotocin-diabetic male rats has been studied. Both phenformin and tolbutamide mimic the action of insulin in stimulating hepatic steroid metabolism in a dose-dependent manner in control cells. Unlike insulin, however, both drugs give a similar effect in cells derived from diabetic animals although to a lesser extent. Both drugs can partially restore the effect of insulin in cells derived from diabetic animals. Biguanides and sulphonylureas, therefore, have a direct effect on liver cells to mimic insulin action and can still have an effect under conditions where insulin is inactive. Both types of oral hypoglycaemics can also affect insulin-insensitive cells isolated from diabetic rat liver to restore to a certain extent their response to insulin.     

The effects of valproate on intermediary metabolism in isolated rat hepatocytes and intact rats

Valproate is a valuable anticonvulsant which is associated with hepatotoxicity in some patients. In concentrations in the range found in man during valproate therapy (0.1-1.0 mM), it inhibited pyruvate and palmitate oxidation, urea synthesis and gluconeogenesis by 30-50% in isolated rat hepatocytes. Valproate (100 mg/kg body weight) is also hypoglycaemic and hypoketonaemic in fasted rats. All these inhibitions can be explained in terms of the accumulation of valproyl-CoA and its further metabolites in the matrix of hepatic mitochondria. Although these inhibitions are only partial, and normally well tolerated, they could significantly impair liver function when there is an additional insult, such as may occur with multiple drug therapy or if there is already an inborn error of metabolism. Such an association with inborn errors may explain the higher incidence of valproate-associated toxicity in children. It may be of more value to measure blood urea and ammonia concentrations routinely shortly after starting valproate therapy than to do conventional liver function tests.     

Effects of two newly synthesized 4-hydroxycoumarin derivatives (4-OHC) on isolated rat hepatocytes

To clarify the hepatotoxicity of two newly synthesized derivatives of 4-hydroxycoumarin (OX and AC) with proven anticoagulant activity in comparison with warfarin, we investigated freshly isolated rat hepatocytes. Hepatocyte damage after incubation with OX, AC and warfarin at a final concentration of 1 x 10(-8) M to 1 x 10(-3) M was assessed by measuring cell viability, lactatdehydrogenase (LDH) activity and glutathione (GSH) levels. The results of cell viability assessment showed that warfarin had the highest toxicity, followed by OX and AC. LDH activity of the tested compounds was mostly increased by warfarin. According to the average effective concentration of the compounds on this parameter, warfarin possessed the most significant toxic effect (EC50 = 1 x 10(-7) M), followed by AC (EC50 = 9.7 x 10(-5) M) and OX (EC50 = 5.0 x 10(-4) M). The observed cytotoxic effects were most pronounced in the highest concentration 1 x 10(-3) M as follows: warfarin, AC and OX. The differences in the effects of OX and AC may be explained by the differences in the electronic structure of the novel compounds, as assessed by molecular modeling.     

Cytotoxic and immunomodulatory effects of polyhydroxyoctane isolated from Lentinus polychrous mycelia

A polyhydroxyoctane, 6-methylheptane-1,2,3,4,5-pentaol (MHP), was first isolated from mycelia of the Thai edible mushroom Lentinus polychrous. MHP was evaluated for its cytotoxic and immunomodulatory effects in vitro. MHP was slightly cytotoxic to murine splenocytes but not to RAW264.7 cells or peripheral blood mononuclear cells. MHP decreased nitric oxide and intracellular O₂ ^? production from lipopolysaccharide- and phorbol-12-myristate-13-acetate-activated RAW264.7 cells at levels of 78.98 ± 4.72 and 78.48 ± 2.41 % of controls, respectively. The mRNA expression of pro-inflammatory mediators, including iNOS, TNF-α, IL-1β, IL-6, COX-1 and COX-2, were significantly suppressed by MHP. In addition, MHP significantly increased the proliferation of phytohemagglutinin and pokeweed mitogen-induced splenocytes. These results indicate that MHP is able to modulate inflammatory responses and the proliferation of both T- and B-lymphocyte cells, suggesting that MHP may be a good natural immunomodulator.     

Cytotoxic effects of mansonone E and F isolated from Ulmus pumila

Two sesquiterpenoids, mansonone E (ME) and mansonone F (MF) were first isolated from the dried root bark of Ulmus pumila (shironire in Japanese), and their antiproliferative activities on human tumor cells were evaluated in vitro. ME had more potent cytotoxic effects on four tumor cell lines, human cervical cancer HeLa, human malignant melanoma A375-S2, human breast cancer MCF-7, and human histiocytic lymphoma U937, than those of MF. The results showed that ME induced oligonucleosomal fragmentation of DNA in HeLa cells and activated caspase-3, followed by the degradation of the inhibitor of caspase-activated DNase, decreased the expression of anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-(XL), and increased that of proapoptotic Bax.     

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.     

Stereoselective effects of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) on schedule-controlled behavior

The effects of R(?)?, S(+) and R, S-1-2-5-dimethoxy-4-methylphenyl)-2-Aminopropane (DOM) were studied using rats responding under a fixed interval two-min schedule of food presentation. All three drugs decreased average rates of responding in a dose-related manner, with R-DOM being five to six times more potent than S-DOM but only about 1.2 times more potent than R,S-DOM. Relatively high doses of R,S-DOM and S-DOM increased the low response rates occurring at the beginning of the fixed interval and decreased the higher response rates occuring at the end of the interval (rate-dependent effects). These results are discussed in terms of the stereoselective metabolism of DOM and of the structural similarities between R-DOM and the behaviorally active isomer of LSD.