Effect of Antihepatotoxic Agents Against Microcystin-LR Toxicity in Cultured Rat Hepatocytes

Primary cultures of adult rat hepatocytes were used to investigate the effects of two putative therapeutic agents, dithioerythritol and silymarin on microcystin-LR-induced hepatotoxicity. Cell injury was assessed by the extent of cellular [¹⁴C]adenine nucleotides and lactate dehydrogenase (LDH) release into the medium and the extent of hepatocyte detachment from monolayers. Microcystin-LR (1 µM) induced a significant release of both ¹⁴C-labeled nucleotides and LDH from hepatocytes as well as significant detachment of cells from monolayers. Although both dithioerythritol (0.63–5 mM) and silymarin (25–200 µM) reduced the amount of marker release and cell detachment from microcystin-LR-treated wells, silymarin provided significantly greater protection than dithioerythritol at one-tenth the concentration. Furthermore, silymarin and dithioerythritol treatment prevented morphological deformations and detachment of cells.     

原代培养大鼠肝细胞中观察十种中药的抗肝毒作用

利用半乳糖胺ＧａｌＮ或四氯化碳ＣＣＩ４引起原代培养大鼠肝细胞损伤模型,研究１０种中药水提液的抗肝毒作用。结果表明：丹参、田基黄、过路黄、野萄花２—５０ｍｇ／ｍｌ;女贞子、车前子１０—５０ｍｇ／ｍｌ;山栀、垂盆草、海金沙及干姜５０ｍｇ／ｍｌ可使含ＧａＩＮ５ｘ１０－３ｍｏｌ／Ｌ肝细胞培养液中ＧＰＴ活性显著降低。其中丹参与田基黄又分别在ＣＣＩ４１．０ｘ１０－２ｍｏｌ／Ｌ损伤肝细胞培养液及ＣＧＩ４实验性肝损伤小鼠中验证,均显示明显的护肝作用．     

齐墩果酸抗环磷酰胺所致大鼠肝细胞损伤作用

目的研究齐墩果酸（OA）对环磷酰胺所致大鼠肝细胞损伤的保护作用。方法分离大鼠肝细胞，进行原代培养。检测实验大鼠肝细胞上清液中丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）和肝细胞MTT值。结果环磷酰胺可使肝细胞上清液ALT、AST及LDH活力升高，肝细胞MTT值减小，齐墩果酸能抑制上述变化。结论齐墩果酸可抗环磷酰胺所致肝细胞损伤。     

Proliferation of Hepatocytes and Attenuation from Carbon Tetrachloride Hepatotoxicity by Gadolinium Chloride in Rats

Abstract: Intravenous injection of gadolinium chloride (GdCl₃) at a dose of 10 mg/kg caused an increase in proliferating cell nuclear antigen labeling index and the grade of pyronin positivity (RNA level) in rat liver. In CCl₄-exposed rats, pretreatment with GdCl₃ also showed a preventive effect of the liver injury both biochemically and histologically. Moreover, the proliferative action preceded the attenuative effect of the liver injury. Results suggest that GdCl₃ induces hepatocyte proliferation, and this action of GdCl₃ may modify the development of CCl₄-induced liver injury.     

Evaluation of the Uptake of Pravastatin by Perfused Rat Liver and Primary Cultured Rat Hepatocytes

Purpose. We have already demonstrated that the HMG-CoA reductase inhibitor, pravastatin is actively taken up by isolated rat hepatocytes via a multispecific anion transporter (Yamazaki et al., Am. J. Physiol. 264, G36-44, (1993)). We further attempted the quantitative evaluation of this uptake in different experimental systems. Methods. We have quantified the initial uptake of pravastatin by both primary cultured hepatocytes and by isolated perfused rat liver using the multiple indicator dilution (MID) method. Results. The permeability surface area product for the influx (PSinf) of pravastatin evaluated in MID study was comparable with those reported previously in isolated rat hepatocytes and in vivo. Furthermore, the highly concentrative uptake (influx clearance >> efflux clearance) of pravastatin was confirmed by kinetic analysis of the dilution curves obtained in the MID study. On the other hand, the uptake by primary cultured cells was significantly lower than that by isolated cells, and the ability of hepatocytes to take up pravastatin showed a decrease with time in culture (0-96 hr). The Vmax for uptake diminished with increasing time in culture, while no significant change was observed in both Km and nonspecific diffusion clearance. Conclusions. The MID method in isolated perfused liver which maintains the spatial and anatomical architecture can be used to quantitatively evaluate the initial uptake of pravastatin. Furthermore, the ability of hepatocytes to take up pravastatin is diminished in culture with time and this is caused by a decrease in Vmax.     

Inhalation Carcinogenicity and Chronic Toxicity of Carbon Tetrachloride in Rats and Mice

Carcinogenicity and chronic toxicity of carbon tetrachloride were examined by inhalation exposure of 50 F344 rats and 50 BDF1 mice of both sexes to carbon tetrachloride at 0 (clean air), 5, 25, or 125 ppm (v/v) for 6 h/day, 5 days/wk, for 104 wk. Incidences of hepatocellular adenomas and carcinomas in rats and mice of both sexes and of adrenal pheochromocytomas in mice of both sexes were significantly increased dose-dependently. Hepatocellular carcinomas and cirrhosis significantly occurred in the 125-ppm-exposed rats of both sexes, and 3 cases of hepatocellular carcinomas and increased incidences of hepatic altered cell foci were noted in the 25-ppm-exposed female rats. Hepatocellular carcinomas were induced in mice of both sexes at 25 and 125 ppm, and hepatocellular adenomas occurred in females at 5 ppm without any degenerative or necrotic change in hepatocytes. Hepatocellular carcinomas metastasized to the lung. The chronic hepatotoxicity was characterized by cirrhosis, fibrosis, and fatty change in rats, and ceroid deposition, bile-duct proliferation, and hydropic change in mice. Survival rates were decreased in the 125-ppm-exposed rats and mice of both sexes and in the 25-ppm-exposed female mice, in association with decreased body weights. The decreased survival rates were considered to be causally related to both various tumors including hepatocellular carcinomas and severe chronic progressive nephropathy in rats and to hepatocellular carcinomas in mice. This study provided clear evidence of carcinogenicity for carbon tetrachloride in rats and mice. A cytotoxic-proliferative and genotoxic mode of action for carbon tetrachloride-induced hepatocarcinogenesis was suggested.     

Effect of Rat Serum Lipoproteins on mRNA Levels and Amiodarone Metabolism by Cultured Primary Rat Hepatocytes

Hyperlipidemia can significantly increase amiodarone (AM) in vivo liver uptake and decrease its velocity of microsomal metabolism. Here, hepatocytes isolated from normolipidemic (NL) and hyperlipidemic rats were incubated with AM in the presence or absence of diluted NL or hyperlipidemic serum. The serum was added either as preincubation before drug, or concurrently with drug; incubations without rat serum were used as controls. The hepatocyte levels of mRNA for several proteins and enzymes were also measured. Disappearance of AM was seen up to 72 h. There was little difference between hepatocytes from NL or hyperlipidemic animals in intrinsic clearance (CL_int) of AM. The effect of hyperlipidemic rat serum, either before or with AM, was profound, causing a significant reduction in the CL_int. Reductions were seen in mRNA for cytochrome P450 1A1, 3A2, and 2D1, some transporters, and low‐density lipoprotein receptors after exposure of hepatocytes to lipoprotein‐rich sera. In conclusion, exposure of isolated hepatocytes to hyperlipidemic serum caused decreases in AM CL_int and lower mRNA levels for some proteins involved in the uptake and metabolism of AM. When coincubated with serum, an additional effect of increased binding to lipoproteins seemed to further contribute to a reduced CL of AM.     

Mechanism of Protection Against Carbon Tetrachloride Toxicity. I. Prevention of Lethal Effects by Partial Surgical Hepatectomy

ABSTRACT

Both partial surgical hepatectomy and a challenge with a small dose of CC₁₄ depress the metabolism of xenobiotics in the liver. Infact, hepatocytes become provided with metabolic activity rdtes which are peculiar of either embryo or newborn rat lver.

These experiments have shown that partial surgical hepatectomy prevents rats from death caused by otherwise lethal doses of CC₁₄. At the same time, sham-operated animals survive to a limited ex= tent after a large dose of the halogenocompound.

Investigations carried out on the metabolic efficiency of liver microsomes, both in vitro and in vivo, clearly demonstrate that the preventive effect against CC₁₄ depends mainly on the impaired metabolic activity of endoplasmic reticulum.     

Effects on Lipid Peroxidation and Antioxidative Enzymes of Euonymus alatus in Cultured Rat Hepatocytes

Abstract: The Euonymus alatus (Thunb.) Sieb. has long been used as a crude drug. In this paper, we investigate the effects of E. alatus on cultured hepatocyte cell system and lipid peroxidation in hydrogen peroxide (H₂O₂) treatment conditions. The study covers the physiological activity (the antioxidative activity and the nitrite‐scavenging effect) of E. alatus. H₂O₂ that can produce intracellular free radical was used for inducer of the peroxidation of cellular lipids. Treatment of E. alatus attenuated in cell killing enhanced by increasing concentrations of H₂O₂. The increased malondialdehyde level induced by H₂O₂ treatment was reduced by pre‐treatment of E. alatus. Furthermore, addition of E. alatus in cell culture medium significantly reduced cell killing and content of intracellular antioxidants. Changes in nitrite‐scavenging effect of E. alatus at various concentrations (5–25 mg/ml) and various pH levels (pH 1.2, 4.2 and 6.0) were also observed. The present study was also done to investigate the effects of E. alatus on cultured hepatocyte cell system, H₂O₂‐induced cytotoxicity and antioxidative enzyme activities, including catalase, superoxide dismutase, glutathione peroxidase and glutathione S‐transferase in H₂O₂ treatment conditions. E. alatus treatment had significant protective or elevating activities on these antioxidative enzyme activities compared to a normal group. The results indicate that E. alatus provides a strong antioxidant protection of cells against H₂O₂‐induced oxidative stress.     

The Effect of an 11.5-hr/Day Exposure Schedule on the Distribution and Toxicity of Inhaled Carbon Tetrachloride in the Rat

The Effect of an 11.5-hr/Day Exposure Schedule on the Distributionand Toxicity of Inhaled Carbon Tetrachloride in the Rat. PAUSTENBACH,D. J., CHRISTIAN, J. E., CARLSON, G. P., AND BORN, G. S. (1986),Fundam. Appl. Toxicol. 6,472–483. This study evaluatedthe differences in toxicity and tissue distribution for 16 groupsof male rats repeatedly exposed to 100 ppm of ¹⁴CCl₄ vaporsfor 8 or 11.5 hr/day for periods of 1 to 10 days. Serum sorbitoldehydrogenase (SDH) was also evaluated for its sensitivity atdetecting CCl₄-induced hepatotoxicity. Following 1, 2, 3, 4,5, 7, 11, and 14 days, one group of rats from each exposureschedule was sacrificed and ¹⁴C activity in seven tissues andserum SDH levels were measured. To compare the effects of CCl₄on the liver and kidney following repeated exposure to the twoschedules, one group of rats was exposed for 8 hr/day for 10of 12 consecutive days and another for 11.5 hr/thy for 7 of12 consecutive days so that each group received essentiallythe same dose (8000 ppm-hr) of CCl₄. The 11.5- hr/day exposureschedule, compared to rats exposed 8-hr/day, produced minorchanges in the distribution and concentration of ¹⁴C (CCl₄ equivalents)in various tissues. Following 1 and 2 weeks of exposure to eitherschedule, the fat, liver, lungs and adrenals had the highestconcentration of CCl₄ equivalents. There were no significantdifferences in CCl₄-induced hepatotoxicity or nephrotoxicitybetween rats exposed to the two schedules following either 1or 2 weeks of exposure as measured by histopathology. In contrast,rats exposed 11.5 hr/day had significantly higher SDH levelsthan those exposed to the 8-hr/thy schedule; thus suggestingthat the 11.5-hr schedule did produce a measurably greater degreeof hepatotoxicity, although it was too subtle to detect pathologically.Rats exposed for a fourth and fifth thy during the second weekof the 11.5-hr schedule had a significantly greater concentrationof ¹⁴C activity in the fat than rats exposed to the 8-hr/dayschedule as well as severe fatty infiltration of the liver andhigher serum SDH activity. This study demonstrated that SDHis a very useful assay for detecting subtle changes in liverinjury as compared to histopathology. It was also shown thateven at relatively low vapor concentrations, modest changesin dosage regimen, like those involving unusual (e.g., 10- orl2-hr/ day) work schedules, can have a measurable effect onthe distribution of the chemical and the degree of toxicity.These results support published recommendations which suggestthat for persons working long shifts, occupational exposurelimits for systemic toxicants with half-lives between 4 and200 hr be reduced so that these persons will have the same marginof safety from any adverse effects.     

Effect of Cryopreservation on Enzyme and Transporter Activities in Suspended and Sandwich Cultured Rat Hepatocytes

Freshly-isolated rat hepatocytes are commonly used as tools for hepatic drug disposition. From an ethical point of view, it is important to maximize the use of isolated hepatocytes by cryopreservation. The present study compared overall hepatocyte functionality as well as activity of the organic anion transporting polypeptide (Oatp), multidrug resistance-associated protein 2 (Mrp2), and UDP-glucuronosyltransferase 1 (Ugt1), in in vitro models established with cryopreserved and freshly-isolated hepatocytes. A similar culture time-dependent decline in cellular functionality, as assessed by urea production, was observed in sandwich-cultured hepatocytes (SCH) obtained from freshly-isolated and cryopreserved cells. Concentration-dependent uptake kinetics of the Oatp substrate sodium fluorescein in suspended hepatocytes (SH) or SCH were not significantly affected by cryopreservation. Mrp2-mediated biliary excretion of 5 (and 6)-carboxy-2′,7′-dichlorofluorescein by SCH was assessed with semi-quantitative fluorescence imaging: biliary excretion index values increased between day 3 and day 4, but did not differ significantly between cryopreserved and freshly-isolated hepatocytes. Finally, telmisartan disposition was evaluated in SCH to simultaneously explore Oatp, Ugt1, and Mrp2 activity. In order to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation, a mechanistic cellular disposition model was developed. Basolateral and canalicular efflux as well as glucuronidation of telmisartan were affected by cryopreservation. In contrast, the disposition parameters of telmisartan-glucuronide were not impacted by cryopreservation. Overall, the relative contribution of the rate-determining processes (uptake, metabolism, efflux) remained unaltered between cryopreserved and freshly-isolated hepatocytes, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes when studying these cellular disposition pathways.     

Partial Maintenance of Taurocholate Uptake by Adult Rat Hepatocytes Cultured in a Collagen Sandwich Configuration

Purpose. This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. Methods. Hepatocytes isolated from male Wistar rats (230–280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein, Na⁺/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. Results. By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na⁺-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 M) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent K_m values were comparable for day-0 and day-5 hepatocytes (32–41 M). In contrast, the V_max values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. Conclusions. A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.     

Decreasing Carbon Tetrachloride Toxicity using Date-seed (Phoenix dactylifera L.) Steeping in Rats

Objective

Many toxic compounds in foods cause liver damage and disturbance of bodily function. Inflammation will precede liver damage as an initial response to poisoning. The inflammatory response depends heavily on the strength of the body’s immune system. Many foods, drugs, and other compounds can decrease the immune system, but few serve as immunostimulants. This study aims to prove the decreasing of carbon tetrachloride toxicity using date-seed (Phoenix dactylifera L.) steeping to improve rat immunity.

Methods

This was an experiment with pre- and posttest with a control group design. Wistar white rats were grouped into 6 groups, healthy control (HC), negative control (NC), positive control (PC), treatment dose 1 g/ kg (T1), treatment dose 3 g/kg (T3), and treatment dose 5 g/kg (T5). All of the groups were induced by CCL₄ before treatment except the HC group. The observed data were interleukin-6 (IL-6), lymphocyte count, and C-reactive protein (CRP). Data from the groups were compared with an ANOVA test and followed by a post hoc test if a significant result was found.

Results

The results showed that there were significant differences between IL-6, lymphocyte count, and CRP between HC and other groups that CCL₄-induced. After the delivery of date-seed steeping, levels of IL-6 and CRP decreased, and the lymphocyte count increased significantly. The group with the 5 g/kg treatment dose was the most effective group for inhibiting the increase of IL-6 and CRP, but a dose of 3 g/kg was the most effective to increase lymphocyte count.

Conclusion

Date-seed steeping suppresses pro-inflammation mediators; it has a potency which improves cytokine pro-inflammation by inhibiting the inflammation process. Thus, date seed can be used as a cheap source of anti-inflammation that can be considered as a health opportunity for developing countries.

     

Potentiation of Chlorinated Hydrocarbon Toxicity by 2,5-Hexanedione in Primary Cultures of Adult Rat Hepatocytes

Potentiation of Chlorinated Hydrocarbon Toxicity by 2,5-Hexanedionein Primary Cultures of Adult Rat Hepatocytes. Jernigan, JamesD., Pounds, Joel G. and Harbison, Raymond D. (1983). Fundam.Appl. Toxicol. 3:22-26. Primary cultures of adult rat hepatocyteswere used to investigate potentiation of halocarbon-inducedhepatotoxicity by aliphatic ketones. Male Sprague-Dawley ratswere pre-treated with corn oil or 2,5-hexanedione (HD; 15 mmol/kg,po) in corn oil. Eighteen hours later the hepatocytes were isolatedand cultured in Williams' Medium E and exposed to several concentrationsof the hepatotoxicants carbon tetra-chloride, chloroform, deutero-chloroforra,or 1,1,2-trichloroethane. The cytotoxicity of these halocarbonsas measured by release of the cytosolic enzyme lactate dehydrogenaseinto the culture medium was both time- and concentration dependent.Halocarbon-induced cytotoxicity was exacerbated in cells isolatedfrom HD-pretreated rats with significant increases in LDH releaseover cells isolated from corn oll-pretreated rats. In addition,chloroform was significantly more toxic than deutero-chloroformin hepatocytes from either corn oil- or HD-pretreated rats.Primary monolayer cultures were useful for studying ketone-inducedpotentiation, halocarbon-induced hepatocellular toxicity, andthe mechanisms by which these effects occur.     

Effect of Purified Saponin Mixture from Astragalus corniculatus on Toxicity Models in Isolated Rat Hepatocytes

A purified saponin mixture (PSM) isolated from Astragalus corniculatus Bieb. (Fabaceae) was investigated for its protective effect in two models of toxicity, carbon tetrachloride (CCl₄) and tert-butyl hydroperoxide (t-BuOOH), using isolated rat hepatocytes. CCl₄ undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (CCl₃) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. Oxidative damage is widely recognized as being involved in the development of many pathological conditions. In our experiment, t-BuOOH was used as a model of oxidative stress. The hepatocytes were incubated with the PSM alone (0.01–100 μ M) and along with CCl₄ (86 μ M) and t-BuOOH (75 μ M). As a sign of cytotoxicity, cell viability was used. CCl₄ and t-BuOOH significantly decreased hepatocyte viability. Our data indicate that PSM showed lower toxic effects compared to CCl₄ and t-BuOOH and in combination exerted statistically significant protection of cell viability against the toxic agents.     

The Effect of Nicotine and Its Interaction with Carbon Tetrachloride in the Rat Liver

Abstract: In order to study the effects of nicotine on liver, groups of rats were given nicotine doses that simulated those seen in chronic smoking (54 and 108 μmol/l of nicotine) for 10 days. A subgroup was also given a single subcutaneous injection of 6 g/kg of carbon tetrachloride (CCl₄) shortly before the animals of the group were killed. Histology demonstrated a significant hepatotoxic effect in the group receiving 108 μmol/l of nicotine when compared with the control group in the form of fatty change, focal or confluent necrosis and dark-cell change. The effects in pregnant rats were less severe. Carbon tetrachloride alone induced significant fatty change and focal necrosis in non-pregnant rats but not in pregnant rats. Nicotine also aggravated the CCl₄ induced pathological changes in livers of both non-pregnant and pregnant animals. Thus nicotine alone, when given at a concentration of 108 μmol/l, exerted hepatotoxic effects; the alkaloid also aggravated the hepatotoxicity of CCl₄. Pregnant rats were more resistant to the hepatotoxic effects produced by nicotine and CCl₄.     

Effect of Dosing Vehicle on the Developmental Toxicity of Bromodichloromethane and Carbon Tetrachloride in Rats

Several halocarbons have been shown to cause full-litter resorption(FLR) in Fischer-344 rats when administered orally in corn oil.Since halocarbons often occur as contaminants of drinking water,we sought to determine the influence of the vehicle, aqueousversus lipid, on the developmental toxicity of two of theseagents. In separate assays, bromodichloromethane (BDCM) andcarbon tetrachloride (CCl₄. were administered by gavage to Fischer-344rats on gestation days (GD) 6–15 at 0, 25, 50, or 75 mg/kg/dayin either corn oil or an aqueous vehicle containing 10% EmulphorEL-620. Dams were allowed to deliver and the litters were examinedpostnatally. Uteri of females that did not deliver were stainedwith 10% aminonium sulfide to detect FLR. Effects of both agentson maternal weight gain were slightly more pronounced in theaqueous vehicle at lower doses, but at the highest dose, CCl₄was more maternally toxic in corn oil. Developmentally, bothagents caused FLR at 50 and 75 mg/kg in both vehicles. At 75mg/kg, dams receiving corn oil had significantly higher ratesof FLR (83% for BDCM, 67% for CCl₄) compared to their aqueous-vehiclecounterparts (21% for BDCM, 8% for CCl₄). Blood concentrationsof BDCM following GD-6 gavage revealed a shorter eliminationhalflife in the aqueous dosing vehicle (2.7 h) compared to theoil vehicle (3.6 h). Benchmark doses of CCl₄ were similar forthe oil (18.9 mg/kg) and aqueous (14.0 mg/kg) vehicles. ForBDCM, the corn oil vehicle yielded a less conservative (i.e.,higher) value (39.3 mg/kg) than the aqueous vehicle (11.3 mg/kg),reflecting different confidence intervals around the estimated5%-effect dose levels.     

Method for Studying the Permeability of the Rat Intestinal Tract to Carbon Tetrachloride

Previous inhalation studies with ¹⁴CC1₄ in rats and monkeys have shown significant amounts of unidentified ¹⁴C compound(s) to be excreted in the feces. The isolated gut sac was used to determine if CC1₄ could potentially be eliminated by intestinal exsorption, the direct transfer from blood to the intestinal lumen. Segments of duodenum, jejunum, ileum, and colon were removed, ligated at one end, and placed in a tissue bath filled with buffer containing CC1₄. Serosal and mucosal fluids were analyzed for CC1₄ by gas chromatography. Fick's law of diffusion was used to determine the permeability coefficient of CC1₄. The kinetic analysis is simplified when sink conditions prevail, i.e., the serosal concentration is constant and much greater than the mucosal concentration. There was significant loss of CC1₄ from the serosal buffer due to the need to oxygenate with 95% O₂ 5% CO₂, making accurate determination of permeability impossible. Saturation of the carbogen with CC1₄ resulted in a steady-state concentration in the serosal buffer. The permeability of CC1₄ was similar in all three segments of the small intestine. Permeability in the colon was slightly less than in the small intestine. This method allows for the study of the direct intestinal elimination of volatile solvents.     

Effects of the Amino Acid Constituents of Microcystin Variants on Cytotoxicity to Primary Cultured Rat Hepatocytes

Microcystins, which are cyclic heptapeptides produced by some cyanobacterial species from algal blooms, strongly inhibit serine/threonine protein phosphatase and are known as hepatotoxins. Microcystins have many structural variations, yet insufficient information is available on the differences in the cytotoxic potentials among the structural variants. In this study, the cytotoxicities of 16 microcystin variants at concentrations of 0.03–10 μg/mL to primary cultured rat hepatocytes were determined by measuring cellular ATP content, and subsequently determined by their 50% inhibitory concentration (IC₅₀). Differences in the amino acid constituents were associated with differences in cytotoxic potential. [d-Asp³, Z-Dhb⁷] microcystin-LR exhibited the strongest cytotoxicity at IC₅₀ of 0.053 μg/mL among the microcystin variants tested. Furthermore, [d-Asp³, Z-Dhb⁷] microcystin-HtyR was also highly cytotoxic. These results suggest that both d-Asp and Z-Dhb residues are important in determining the cytotoxic potential of microcystin variants.     

Assessment of the Toxicity of Chemical Mixtures with Isolated Rat Hepatocytes: Cadmium and Chloroform

The suitability of isolated rat hepatocytes for the investigationof the toxicity of chemical mixtures has been studied. Cadmiumchloride and chloroform were used because both had been previouslyinvestigated in hepatocytes and both produce hepatotoxicityafter in vivo administration. The addition of the two chemicalscaused an increase in cytotoxicity as assessed by loss of intracellularpotassiumion and aspartate aminotransferase. There was evena toxic response from the mixture at concentrations where thechemicals alone yielded no such response. The metabolic parameter,lactate to pyruvate ratio, was less consistently affected. Thenature of the interaction could, under the varying conditionsemployed, be described as one of synergism, potentiation, oraddition. The data support the proposed role of isolated hepatocytesin screening chemical mixtures for toxic effects.