Food preservatives sodium benzoate and propionic acid and colorant curcumin suppress Th1-type immune response in vitro

Food preservatives sodium benzoate and propionic acid and colorant curcumin are demonstrated to suppress in a dose-dependent manner Th1-type immune response in human peripheral blood mononuclear cells (PBMC) in vitro. Results show an anti-inflammatory property of compounds which however could shift the Th1–Th2-type immune balance towards Th2-type immunity.     

Effect of sulfite on red blood cell deformability ex vivo and in normal and sulfite oxidase-deficient rats in vivo

The effect of sulfite, a widely used food additive, on red blood cell deformability ex vivo and in vivo was investigated. Ex vivo experiments were conducted in human blood exposed to sulfite (5, 10 and 20 mM). In vivo experiments were carried out in sulfite oxidase-competent (SOXC) and sulfite oxidase-deficient (SOXD) rats. In the in vivo experiments, sulfite was administered in the form of sodium metabisulfite (Na₂S₂O₅, 25 mg/kg/day) via drinking water. Vitamin E dissolved in olive oil at a dose of 50 mg/kg was administered by gastric gavages. Red blood cell (RBC) deformability was determined at various fluid shear stresses using an ektacytometer. Ex vivo sulfite exposure to RBC did not affect RBC deformability. In the in vivo experiments, although RBC deformability was not affected by sulfite treatment in SOXD rats, it was found to be significantly increased in SOXC rats. Vitamin E treatment in combination with sulfite caused impairment in RBC deformability in both SOXC and SOXD rats. We suggest that sulfite needs to be oxidized in order to improve RBC deformability.     

Lack of formic acid production in rat hepatocytes and human renal proximal tubule cells exposed to chloral hydrate or trichloroacetic acid

The industrial solvent trichloroethylene (TCE) and its major metabolites have been shown to cause formic aciduria in male rats. We have examined whether chloral hydrate (CH) and trichloroacetic acid (TCA), known metabolites of TCE, produce an increase in formic acid in vitro in cultures of rat hepatocytes or human renal proximal tubule cells (HRPTC). The metabolism and cytotoxicity of CH was also examined to establish that the cells were metabolically active and not compromised by toxicity. Rat hepatocytes and HRPTC were cultured in serum-free medium and then treated with 0.3–3 mM CH for 3 days or 0.03–3 mM CH for 10 days, respectively and formic acid production, metabolism to trichloroethanol (TCE-OH) and TCA and cytotoxicity determined. No increase in formic acid production in rat hepatocytes or HRPTC exposed to CH was observed over and above that due to chemical degradation, neither was formic acid production observed in rat hepatocytes exposed to TCA. HRPTC metabolized CH to TCE-OH and TCA with a 12-fold greater capacity to form TCE-OH versus TCA. Rat hepatocytes exhibited a 1.6-fold and three-fold greater capacity than HRPTC to form TCE-OH and TCA, respectively. CH and TCA were not cytotoxic to rat hepatocytes at concentrations up to 3 mM/day for 3 days. With HRPTC, one sample showed no cytotoxicity to CH at concentrations up to 3 mM/day for 10 days, while in another cytotoxicity was seen at 1 mM/day for 3 days. In summary, increased formic acid production was not observed in rat hepatocytes or HRPTC exposed to TCE metabolites, suggesting that the in vivo response cannot be modelled in vitro. CH was toxic to HRPTC at millimolar concentrations/day over 10 days, while glutathione derived metabolites of TCE were toxic at micromolar concentrations/day over 10 days [Lock, E.A., Reed, C.J., 2006. Trichloroethylene: mechanisms of renal toxicity and renal cancer and relevance to risk assessment. Toxicol. Sci. 19, 313–331] supporting the view that glutathione derived metabolites are likely to be responsible for nephrotoxicity.     

Moderating influence of the drinking water disinfection by-product dibromoacetic acid on a dithiocarbamate-induced suppression of the luteinizing hormone surge in female rats

The disinfection by-product dibromoacetic acid (DBA) has been found in female rats to increase circulating concentrations of both estradiol (E2) and estrone (E1). This effect is apparently due, at least in part, to a suppression in hepatic catabolism. The present study investigated whether DBA, by increasing sex steroid levels, is able either to augment the hypothalamic up-regulation involved in triggering a luteinizing hormone (LH) surge, or to affect the ability of the neurotoxicant sodium dimethyldithiocarbamate (DMDC) to block the surge. Sprague–Dawley rats were gavaged for 14 days with DBA (0–150 mg/kg) and ovariectomized on dosing day 11, and at the same time implanted with an estradiol capsule to generate daily LH surges. An injection of 0.1 mM/kg DMDC was administered at 13:00 h on day 14 and blood was sampled over the afternoon. DBA induced a dose-related increase in total estrogens. For identified surges, areas under the LH curve partitioned into two groups, comprising the two lower (0 and 37.5 mg/kg DBA) and the two higher (75 and 150 mg/kg) treatment groups. Consequently, low and high DBA groups were compared and found to be significantly different. At 150 mg DBA/0.1 mM DMDC, the timing of an identifiable LH peak was comparable to non-DMDC females, unlike the 37.5 mg DBA/0.1 mM DMDC group in which the appearance of peak concentrations was delayed. A significant effect with DBA treatment alone was not present. Results indicated that this exposure to DBA induced a dose-related increase in total estrogen concentrations that paralleled a diminished DMDC blockade of the LH surge. The effect appeared to be attributable to an augmentation in the estrogen-associated up-regulation in brain mechanisms stimulating the surge.     

Effects of sorbic acid and sorbic acid-nitrite in vivo on bone marrow chromosomes of mice

The effect of sorbic acid alone and in combination with sodium nitrite has been studied on bone marrow chromosomes of mice following 30 days oral treatment. Bone marrow of mice exposed to sorbic acid (15 mg/kg) and sorbic acid nitrite (7.5-1 mg/kg) showed an increase in mitotic index indicating that the drugs had an effect on spindle apparatus. Sorbic acid effected spindle activity but did not damage chromosomes, whereas nitrite itself was clastogenic. However, a combination of half the concentration each of sorbic acid (15 mg/kg) and sodium nitrite (2 mg/kg) together gave synergistic effects, which may be ascribed to the formation of some genotoxic compound in vivo.     

Induction of chronic inflammation in mice treated with titanium dioxide nanoparticles by intratracheal instillation

Titanium dioxide nanoparticles (TNP) are nanomaterials which have various applications including photocatalysts, cosmetics, and pharmaceuticals because of their high stability, anticorrosiveness, and photocatalytic properties. Induction of cytokines and potential chronic inflammation were investigated in mice treated with TNP (5 mg/kg, 20 mg/kg, and 50 mg/kg) by a single intratracheal instillation. Pro-inflammatory cytokines such as IL-1, TNF-a, and IL-6 were significantly induced in a dose-dependent manner at day 1 after instillation. The levels of Th1-type cytokines (IL-12 and IFN-γ) and Th2-type cytokines (IL4, IL-5 and IL-10) were also elevated dose-dependently at day 1 and the inflammatory responses were sustained until the remainder of experimental period for 14 days. By the induction of Th2-type cytokines, the increased B cell distributions both in spleen and in blood, and increased IgE production in BAL fluid and serum were observed. In lung tissue, increase of inflammatory proteins (MIP and MCP) and granuloma formation were observed. Furthermore, the expressions of genes related with antigen presentation (H2-T23, H2-T17, H2-K1, and H2-Eb1) and genes related with the induction of chemotaxis of immune cells (Ccl7, Ccl3, Cxcl1, Ccl4, Ccl2) were markedly increased using microarray analysis. From these data, it could be suggested that TNP possibly cause chronic inflammatory diseases through Th2-mediated pathway in mice.     

Determination of sodium metabisulfite in parenteral formulations by HPIC with suppressed conductivity detection

Sulfurous compounds: sodium sulfite Na2SO3 (E 221), sodium bisulfite NaHSO, (E 222), and sodium metabisulfite Na2S2O5 (E 223) are largely used as antioxidants in many pharmaceutical formulations. A method for determination of sodium metabisulfite in parenteral formulations containing tartrate ions was developed. High-performance ion chromatography (HPIC) method with suppressed conductivity detection was used. A satisfactory separation of SO(3)2- and SO(4)2- was achieved by the proposed HPIC method with 15 mM NaHCO3/0.6 mM Na2CO3 mobile phase and columns with various packing materials: methacrylate polymer -Allsep A-2 Anion (100 x 4.6 mm, 7 microm) and styrene-divinylbenzene copolymer - IonPac AS14A (250 x 4.0 mm, 7 microm). Use of the Allsep A-2 Anion column provides separation of SO(3)2-, SO(4)2- and C4H4O(4)2- present in the investigated products. The calibration plot was linear for 8-267.3 microg/mL sulfite (r = 0.99978, n = 6) and for 3-165.9 microg/mL sulfate (r = 0.9998, n = 6). The limit of detection for SO(3)(2-) and SO(4)2-were 3 microg/mL and 1 microg/mL, respectively.     

Effect of sulfite on macrophage functions of normal and sulfite oxidase-deficient rats.

Sulfite has both an endogenous and an exogenous provenance in the mammalian tissues. The aim of the present study was to assess the effect of sulfite on macrophages functions in normal or sulfite oxidase deficient rats. Rats were divided into eight groups; (1) control group, (2) sulfite group (the rats received sodium meta bi-sulfite (25 mg/kg) in drinking water for 6 weeks), (3) vitamin E group (the rats received Vit E (50 mg/kg) by gavage for 6 weeks), (4) sulfite group+Vit E, (5)sulfite oxidase deficient group (the rats received high-W/Mo-deficient diet. The activity of sulfite oxidase was reduced in rats maintained on the high-W/Mo-deficient diet during the first 21 days of treatment. After the sulfite-oxidase deficiency, the rats continued to receive high-W/Mo-deficient diet for 6 weeks.), (6) sulfite+sulfite oxidase deficient group, (7) Vit E+sulfite oxidase deficient group, and (8) sulfite+Vit E+sulfite oxidase deficient group. Sulfite caused a significant increase in phagocytic and chemotactic activities of peritoneal macrophages. In sulfite-oxidase deficient rats, the increase in phagocytic and chemotactic activities in peritoneal macrophages after sulfite intake was found more than the control rats. Vit E supplementation prevented sulfite induced increase in macrophages functions. These results show that the macrophage functions are sensitive to sulfite intake. The effect of sulfite on macrophage functions may be related to reactive oxygen species. Because Vit E administration was able to modulate significantly sulfite-induced changes in the functions of peritoneal macrophages.     

High-resolution method for regulatory control of Echinacea species in Nutraceuticals by CD-MEKC

One problem in the international regulatory control of Echinacea, a therapeutic Nutraceutical, is recognition of caffeoyl solutes and alkamides in different products. Cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) has been applied to Echinacea spp. in combination with pattern recognition of some caffeoyl solutes. A novel metric based on relative migration time (RMT) data has been developed in CE to address the problem of variable reported migration time.

The CD-MEKC method of Gotti's group using hydroxypropyl-β-cyclodexrin (HP-β-CD; 100 mM) with sodium dodecyl sulphate (SDS; 110 mM), in a triacid background electrolyte (10 mM, pH 8) under 19 kV was adapted to identify two key hydrophilic solutes: chlorogenic acid and cichoric acid present in all commercial products. Two internal markers were taken as reference points to calculate the RMT of any target peak: RMT = t_{m (target)}/t_{m (marker)}.

The RMT method was robust to temperature change from 20 to 40 °C, but sensitive to pH. The lateral shift and reproducibility of the target peak t_{m (target)} were significantly reduced by this novel transformation. In the worst cases migration time variability ranged up to 12% (n = 6); the RMT algorithm reduced this to less than 1%. In general, the RMT transformation reduced the variability of migration time data by a factor of 2–5.

For systematic comparison of electrophoretic profiles for test sample and standard, a new pattern recognition algorithm permits sequential peak-by-peak comparison using specified segments of the electropherograms for comparison of test and Echinacea purpurea (root product) as a standard. This algorithm was capable of rapidly characterising the similarity of target peaks in a test sample relative to those in the reference standard. Combination of the RMT algorithm and pattern recognition in CE is expected to offer a robust approach to international regulatory characterisation and control of Nutraceuticals.     

Sister chromatid exchanges and micronuclei formations induced by sorbic acid and sorbic acid-nitrite in vivo in mice

The in vivo induction of sister chromatid exchanges and micronuclei formations by acute treatment with different concentrations of sorbic acid and by nitrite, individually and in combination, was studied in bone marrow cells of mice. A significant increase in the frequency of sister chromatid exchanges was only observed with the three higher concentrations of sorbic acid when compared to a distilled water control. Sodium nitrite produced a significant increase at all doses tested. A combination of half the concentration of sorbic acid and of sodium nitrite gave an additive effect over that of sorbic acid or sodium nitrite alone. In the micronucleus assay, the highest dose of sorbic acid (150 mg/kg body weight) produced a significant increase in micronuclei formations compared to the distilled water control. Sodium nitrite alone induced significant numbers of micronuclei at all concentrations tested when compared to the negative control. However, a combination of half the concentration of sorbic acid and of sodium nitrite gave synergistic effects which could possibly be ascribed to the formation of certain genotoxic compounds in vivo.     

Cytokine fingerprinting of chemical allergens: species comparisons and statistical analyses

The cellular and molecular mechanisms that result in the induction of chemical respiratory sensitization are unclear, although there is evidence for the development of T helper (Th) 2 type responses and, in some cases, the production of IgE. We have compared cytokine secretion patterns stimulated by topical exposure of BALB/c strain mice or Brown Norway (BN) strain rats to the reference respiratory allergen trimellitic anhydride (TMA), or to the reference contact allergen 2,4-dinitrochlorobenzene (DNCB). Under conditions where TMA and DNCB provoke similar levels of immune activation [increases in lymph node cell (LNC) cellularity and proliferation] divergent cytokine expression patterns are elicited. TMA-activated LNC isolated from BALB/c mice or BN rats elaborated high levels of the Th2-type cytokines interleukin (IL)-10 and IL-13, but relatively little of the Th1-type cytokines IL-12 or interferon γ. For LNC derived from both species there was a requirement for restimulation in vitro with the mitogen concanavalin A for IL-4 production. Generally, DNCB-stimulated LNC displayed the converse type 1 cytokine phenotype. The cytokine secretion profiles of LNC isolated from BN rats were considerably more variable than those observed for LNC from BALB/c mice. Statistically significant differences (P<0.01) between DNCB- and TMA-activated LNC were recorded for all cytokines in BALB/c strain mice. For the BN rat, differences reached statistical significance (P<0.01) only for the expression of IL-4 and IL-13. These data demonstrate that the intrinsic ability of DNCB and TMA to promote preferential Th1- and Th2-type responses, respectively, is species-independent and provide further evidence that chemical respiratory allergens are associated with polarized Th2-type responses. For the prospective assessment of chemical respiratory allergens as a function of induced cytokine secretion profiles, however, these data suggest that the use of the BALB/c strain mouse will provide the more robust method.     

Evaluation of the genotoxic potential of sorbic acid and potassium sorbate.

The genotoxic potential of sorbic acid and potassium sorbate was investigated in vivo and in vitro. Oral administration of sorbic acid (up to 5000 mg/kg body weight) did not induce sister chromatid exchanges or the formation of micronuclei in bone marrow cells of mice. Intraperitoneal treatment of rats with 400-1200 mg potassium sorbate/kg body weight did not alter the elution profile of DNA from isolated liver cells in the in vivo alkaline elution assay. Sorbic acid did not induce DNA repair in cultured human A549 cells in the unscheduled DNA synthesis (UDS) assay. In vitro incubation of the cells with 1-1000 micrograms potassium sorbate/ml, in the absence or presence of rat liver homogenate, did not result in the formation of DNA single-strand breaks in the alkaline elution assay. These results demonstrate that sorbic acid and its potassium salt are not genotoxic in vivo or in vitro. In contrast to sorbic acid and potassium sorbate, sodium sorbate is very sensitive to oxidative degradation; the main oxidation product was identified to be 4,5-oxohexenoate, which was mutagenic in the Ames test.     

Pro-inflammatory and potential allergic responses resulting from B cell activation in mice treated with multi-walled carbon nanotubes by intratracheal instillation

The increased application of engineered carbon nanotubes (CNTs) has also raised the level of public concern regarding possible toxicities caused by exposure to these nanostructures. In this study, pulmonary and systemic immune responses induced by intratracheal instillation of multi-walled carbon nanotubes (MWCNTs) were investigated in mice. Total numbers of immune cells in bronchoalveolar lavage (BAL) fluid were significantly increased in treated groups (5, 20, and 50 mg/kg doses of MWCNTs) and the distribution of neutrophils was elevated at day 1 after instillation. Pro-inflammatory cytokines (IL-1, TNF-α, IL-6, IL-4, IL-5, IL-10, IL-12, and IFN-γ) were also increased in a dose-dependent manner, both in BAL fluid and in blood. Most of the cytokines showed the highest levels at day 1 after instillation and then decreased. Th2-type cytokines (IL-4, IL-5, and IL-10) were elevated in the treated group to levels higher than those of the Th1-type cytokines (IL-12 and IFN-γ). Furthermore, distributions of B cells in spleen and blood were significantly increased at day 1 after instillation, indicating that Th2-type cytokines had activated B cells, causing them to proliferate. Along with the additional numbers of B cells, granuloma formation in the lung tissue and IgE production were also observed, with an intensity dependent on the dose of MWCNTs instilled. Based on these observations, it is suggested that MWCNTs may induce allergic responses in mice through B cell activation and production of IgE.     

Persistent effect of in utero meso-2,3-dimercaptosuccinic acid (DMSA) on immune function and lead-induced immunotoxicity

Meso-2,3-dimercaptosuccinic acid (DMSA) is a drug currently employed for cheltion therapy in lead poisoning; however, little is known about its potential effects on the immune system. To examine the effect of DMSA and its capacity to reverse immunotoxicity resulting from exposure to lead in utero, female Fischer 344 rats were administered lead acetate in drinking water from 2 weeks prior to mating until parturition; DMSA was given by gavage on days 6-21 of gestation. The immune function of the female offspring was tested at 13 weeks of age. The results showed that lead (250 ppm) suppressed Th1-type responses (delayed-type hypersensitivity (DTH), interferon gamma (IFN gamma) production), enhanced a Th2-type response (interleukin-4 (IL-4) production), and increased tumor necrosis factor alpha (TNF alpha) production from macrophages. DMSA treatment (60 mg/kg per day) during pregnancy significantly lowered the blood lead levels of both the embryos and the lactating dams as well as the milk lead level of lactating dams. The chelation treatment also reversed the lead-induced alterations in pup body weight, relative spleen weight, TNF alpha, and IL-4 production. But in utero exposure to DMSA alone resulted in decreased DTH response in adult offspring. This was likely due to a reduced cell recruitment, since plasma monocyte chemoattractant protein-1 (MCP-1) levels were decreased. The DMSA-exposed offspring also demonstrated increased interleukin-2 (IL-2) production. These results suggest that DMSA reverses some of the lead-induced immunotoxicity; however, this treatment itself during embryonic development produces subsequent adult immunomodulation.     

Modulation of Th1/Th2 cytokine production by selective and nonselective phosphodiesterase inhibitors administered to mice

Phosphodiesterase (PDE) inhibitors can modulate the functions of immune cells, including T lymphocytes, due to increased intracellular levels of cyclic nucleotides. The drugs (aminophylline, milrinone and sildenafil) were administered once or five times at 24 h intervals at the following doses: 20 mg/kg, im, 1 mg/kg, im and 1 mg/kg, po, respectively.Th1 and Th2 cytokine levels (IL-2, IFN-γ, IL-4, IL-5, TNF) were determined 12, 24 or 72 h after the last administration of the drugs. A commercial BD^TM Cytometric Bead Array Mouse Th1/Th2 Cytokine Kit (CBA) was used to determine the levels of Th1/Th2 cytokines in the serum.Neither of the PDE inhibitors under investigation administered once changed IFN-γ, TNF and IL-4 production. A single dose of aminophyl-line decreased the production of IL-2 (after 12 h). A single dose of milrinone did not affect Th1/Th2 cytokine secretion. Sildenafil administered once decreased the production of IL-2 (after 72 h). A temporary enhancement in the level of IL-5 was observed 12 h after a single dose of sildenafil. No changes in Th1 and Th2 cytokine production were observed after five doses of PDE inhibitors under investigation. These results indicate that nonstimulated lymphocytes Th1 and Th2 exhibited a slight sensitivity to aminophylline and sildenafil. The drugs under investigation were ineffective inhibitors of Th1/Th2 cytokine production.     

白癜风患者外周血单个核细胞IL-2 IL-10和IFN-γ的表达特点

目的探讨白癜风患者中Th1/Th2型细胞因子表达的情况。方法采用细胞培养技术对白癜风患者和正常对照者的外周血单个核细胞(PBMC)在刀豆素A(ConA)作用下进行体外培养;用酶联免疫吸附试验(ELISA)检测培养上清中白细胞介素(IL)2、IL10,干扰素(IFN)γ含量。结果①白癜风患者PBMC产生IL2水平高于正常对照组(P<0.05),IL10水平低于正常对照组(P<0.01)、IFNγ水平高于正常对照组(P<0.05)。②患者和对照组PBMC加ConA刺激孔中IL2高于相应自然增殖孔(P<0.05,P<0.01)。结论白癜风患者Th1型细胞因子的表达增高,Th2型细胞因子的表达降低,可能在发病机制中起一定作用。ConA可刺激PBMC分泌细胞因子。     

Effects of aspirin-like drugs on canine gastric mucosal blood flow and acid secretion.

1. The effects of aspirin, paracetamol and benorylate were studied on gastric mucosal blood flow (MBF) and acid secretion in canine denervated gastric pouches. 2. Aspirin 20 mM in the unstimulated pouch had no effect; pentagastrin-stimulated acid output, but not MBF, was reduced. Aspirin buffered to pH 6 was ineffective. 3. Aspirin 3-50 mg/kg reaching the pentagastrin-stimulated pouch through the blood, increased acid secretion and MBF, but the MBF:secretion ratio was variably affected. 4. Paracetamol (10 or 20 mg/kg i.v., or 20 mM in the pouch) or benorylate (280 mg/kg orally) mainly had little effect. 5. Circular muscle strips from dog arteries were contracted by prostaglandins E2, F1alpha or F2alpha, and often slightly by indomethacine, but prostaglandin E1 produced variable effects. 6. These results do not favour the view that aspirin causes gastric bleeding in dogs by breakdown of blood vessels due to ischaemia following mucosal vasoconstriction.     

Production of Taxol and its Analogues from Cell Cultures of Taxus wallichiana

Callus cultures of Taxus wallichiana young leaves were established on Gamborg’s (B5) medium supplemented with dicamba (2 mg/l), benzyladenine (0.2 mg/l) and glutamine (292 mg/l). The cell cultures were initiated and maintained on B5 medium supplemented with NAA (1mg/l) and benzyladenine (0.02 mg/l). 0.25 B5 medium supplemented with 2,4-D (2 mg/l) and zeatin (0.5 mg/l) was found to be suitable as production medium. On HPLC analysis using photodiode array detector, the extracts from cell cultures showed the presence of taxanes, viz., taxol, deacetyl baccatin III (DAB) and baccatin III (BA). Precursors and growth retardants significantly improved the production of taxol, DAB and BA. Phenylalanine (1 mM), sodium benzoate (0.2mM), hippuric acid (1 mM) and leucine (1 mM) addition enhanced the accumulation of taxol (6, 4.5, 25 and 2 times, respectively), DAB (7, 9.5, 7.5 and 3 times, respectively) and BA (36, 16, 57 and 2 times, respectively) in cultures grown in production medium. Ancymidol (50µM) and 2-chloroethyl phosphonic acid (50µM) addition has shown significant increase in bioproduction of taxol (3 and 11 times, respectively), DAB (3 and 1.5 times, respectively) and BA (16 and 9 times, respectively). However, chlorocholine chloride (1mM) improved the production of DAB (2.5 times) and taxol (1.7 times) only.     

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24 h of APAP treatment starting at 24 h after seeding (EC₅₀: 3.8 mM, 7.6 mM, and 28.2 mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC₅₀ values were 6.0 mM, 12.5 mM, and 18.8 mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.