Comparison of the toxicity of allyl alcohol,coumarin and menadione in precision-cut rat,guinea-pig,Cynomolgus monkey and human liver slices

 The toxicity of allyl alcohol, coumarin and menadione has been studied in precision-cut liver slice cultures. Liver slices were prepared from male Sprague- Dawley rats, male Dunkin-Hartley guinea-pigs and from samples of Cynomolgus monkey and human liver using a Krumdieck tissue slicer. The liver slices were cultured with the test compounds for 24h in a dynamic organ culture system. Toxicity was assessed by measurement of protein synthesis, potassium content and the MTT assay. At the concentrations examined, menadione produced marked toxicity in liver slices from all four species, whereas rat liver slices were less susceptible to allyl alcohol toxicity. Coumarin produced concentration-dependent toxic effects in rat and guinea-pig liver slices, whereas Cynomolgus monkey and human liver slices were relatively resistant, especially at low coumarin concentrations. At some concentrations of the test compounds examined, the MTT assay appeared to be a less sensitive indicator of toxicity than either protein synthesis or potassium content. These results demonstrate the usefulness of precision-cut liver slices for assessing species differences in xenobiotic-induced toxicity. Received: 21 March 1996/Accepted: 4 June 1996     

Metabolic and structural viability of precision-cut rat lung slices in culture

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72 h. 2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with beta-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600 microm. 3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8 h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72 h incubation. 4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24h; longer incubations did not result in further leakage. 5. It is concluded that precision-cut rat lung slices, of 600 microm thickness, can be maintained metabolically viable in culture for some 8 h.     

Ascorbic acid and paraquat: Oxygen depletion with concurrent oxygen activation

Ascorbic acid and paraquat produce an efficient redox pair which will deplete oxygen from physiological buffer systems. This reaction is partially blocked by superoxide dismutase or catalase and is potentiated by the hydroxyl radical scavenger, dimethyl sulfoxide. Mitochondria isolated after incubation of rat lung slices with 1.0 mm paraquat and 10.0 mm ascorbate were unresponsive to ADP (were “uncoupled”). Also, metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose was inhibited by 50% in lung slice preparations. These results suggest a synergistic interaction of ascorbate and paraquat which results in disruption of subcellular energy metabolism. Paraquat accumulation into lung slices, an active transport process of the pulmonary cell membrane, was also inhibited by the addition of ascorbate. These results suggest that the previously reported in vivo potentiation of paraquat toxicity by ascorbate may be related to either: (1) a decreased subcellular oxygen availability, or (2) the presence of activated oxygen species, or (3) both.     

The use of precision-cut rat lung slices for studying PGF(2α) metabolism

The suitability of a dynamic lung slice culture system as an in vitro model for studying pulmonary metabolism of PGF_2α was assessed. [³H]Prostaglandin F_2α ([³H]PGF_2α), a twenty carbon fatty acid that contains a five-carbon ring and is known to be metabolized by lung in vivo, was incubated with precision-cut rat lung slices in 1.7 ml of Waymouth's buffer fortified with 10% fetal calf serum. At 0, 2, 4 and 8 h after addition of [³H]PGF_2α (1.82 ng/μCi), incubation was stopped and the contents of each vial were analyzed for [³H]PGF_2α and its metabolites using reversed-phase HPLC with radiochemical detection. PGF_2α was metabolized to 15-keto PGF_2α, 13,14-dihydro-15-keto PGF_2α, and two unknown minor polar metabolites. These results indicate that PGF_2α was metabolized in lung slices pathways similar to those seen in vivo. Slice viability was assessed by protein synthesis and light microscopic examination of lung slices through 24 h of incubation. Protein synthesis was maintained and no tissue necrosis was observed over the entire 24 h incubation, indicating that the lung slices were viable for at least 24 h. These results indicate that the dynamic lung slice culture system is an appropriate in vitro model for studying the pulmonary metabolism of PGF_2α.     

Potentiation of the cell specific toxicity of paraquat by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Implications for the heterogeneous distribution of glutathione (GSH) in rat lung

In order to study oxidative stress in the lung, we have developed a rat lung slice model with compromised oxidative defences. Lung slices with markedly inhibited glutathione reductase activity (approximately 80% inhibition) were prepared by incubating slices, with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 microM) in an amino acid-rich medium for 45 min at 37 degrees. These lung slices had similar levels of GSH and ATP and polyamine uptake (a marker of alveolar epithelial type I and II cell function) to control rat lung slices. We have utilized these BCNU pretreated slices to study the effects of the herbicide, paraquat, in comparison to those of 2,3-dimethoxy-1,4-naphthoquinone, a potent redox cycler. Paraquat (10-100 microM) caused only minimal changes in the levels of GSH or ATP in control or compromised slices. In contrast, 2,3-dimethoxy-1,4-naphthoquinone caused a decrease in GSH in control slices but a markedly enhanced decrease in both GSH and ATP in compromised slices. Both compounds had only limited effects on putrescine and spermidine uptake in control slices. However, they caused a marked inhibition in compromised slices. Paraquat had little effect on 5-hydroxytryptamine uptake (a marker of endothelial cell function) in either control or compromised slices whereas the quinone inhibited uptake in the compromised slices. Thus, the lack of effect of paraquat on GSH and ATP does not support the involvement of oxidative stress in its toxicity. In contrast, using polyamine uptake, as a functional marker of alveolar epithelial cell damage, suggests a role for redox cycling. As paraquat is known to be accumulated primarily in alveolar type I and II cells (a small fraction of the lung cell population), our data suggest that only a small proportion of pulmonary GSH and ATP is present in alveolar epithelial type I and II cells but that much larger amounts may be present in endothelial cells. These studies highlight the problem of gross tissue measurements in heterogeneous tissues such as the lung.     

An evaluation of the redox cycling potencies of paraquat and nitrofurantoin in microsomal and lung slice systems

The redox cycling abilities of the pulmonary toxins paraquat and nitrofurantoin have been compared with those of the potent redox cyclers, diquat and menadione in lung and liver microsomes by using the oxidation of NADPH and consumption of oxygen. The relative potencies of these compounds to undergo redox cycling were in the order: diquat approximately menadione much greater than paraquat congruent to nitrofurantoin. This was partly attributed to the much lower affinity (Km) of lung and liver microsomes for paraquat and nitrofurantoin than for diquat and menadione. The potential to redox cycle was assessed in an intact cellular system by determining the oxygen consumption of rat lung slices in the presence (10(-6), 10(-5) and 10(-4) M) or absence of each of the four substrates. At concentrations of paraquat (10(-5) M) known to be accumulated by lung slices, a small but significant stimulation of lung slice oxygen uptake was observed. Nitrofurantoin (10(-4)-10(-6) M) did not affect lung slice oxygen uptake in lung slices, an observation consistent with its being a poor redox cycling compound, which is not actively accumulated into lung cells. This data has important implications in assessing the risk of exposure to paraquat. Low levels of paraquat would not be expected to cause lung damage because insufficient compound is present in the lung to exert its toxicity by redox cycling (due to the high Km observed).     

The effect of chlorpromazine on the uptake and efflux of paraquat in rat lung slices

The uptake of paraquat by rat lung slices was inhibited by chlorpromazine in a concentration- and time-dependent manner. In addition, the efflux of paraquat from lung slices was enhanced by chlorpromazine in a similar fashion. These in vitro findings suggested that chlorpromazine might be useful in vivo in reducing pulmonary paraquat content and, in turn, its pneumotoxicity. However, chlorpromazine potentiated the lethal toxicity of paraquat rather than ameliorating it. This potentiation by chlorpromazine was found to correlate with a reduction in the urinary excretion of paraquat and concomitant increase in pulmonary paraquat concentrations.     

Effects of paraquat,dinoseb and 2,4-D on intracellular calcium and on vasopressin-induced calcium mobilization in isolated hepatocytes

 The effects of the herbicides paraquat, dinoseb and 2,4-D on intracellular Ca²⁺ levels and on vasopressin-induced Ca²⁺ mobilization were investigated in intact isolated hepatocytes. Incubation of rat hepatocytes with paraquat (5 mM for 60 min) and dinoseb (10 μM) resulted in a time-dependent loss of viability by approximately 25%. Viability of cells treated with 2,4-D decreased significantly, dropping to about 20% at 10 mM and 60 min incubation. Exposure of hepatocytes to paraquat (1–10 mM) for 60 min had no effect on the basal level of [Ca²⁺] _i . Additionally, exposure to paraquat had no effect on the magnitude and on the duration of the [Ca²⁺] _i response to vasopressin. In the presence of 2,4-D (1–10 mM), basal [Ca²⁺] _i increases as a function of herbicide concentration. The magnitude of the Δ[Ca²⁺] _i response decreases from 256±8 nM in control to 220±5 nM, at 10 mM 2,4-D. Exposure of hepatocytes to dinoseb (1–10 μM) had no effect on the basal level of [Ca²⁺] _i . However, a strong concentration-dependent decrease in the magnitude of Δ[Ca²⁺] _i in response to vasopressin was noticed at 60 min incubation. Dinoseb markedly inhibited the stimulation of the production of inositol phosphates by vasopressin stimulus. The present study demonstrates that paraquat, 2,4-D and dinoseb cause cell death in hepatocytes by mechanisms not related to an early increase in [Ca²⁺] _i . Additionally, it has been shown for the first time that dinoseb disturbs the transduction mechanism promoted by vasopressin by inhibiting the formation of IP₃. Received: 11 October 1994/Accepted: 5 December 1994     

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.     

Effect of some indole derivatives on xenobiotic metabolism and xenobiotic-induced toxicity in cultured rat liver slices.

In this study the effect of some indole derivatives on xenobiotic metabolizing enzymes and xenobiotic-induced toxicity has been examined in cultured precision-cut liver slices from male Sprague-Dawley rats. While treatment of rat liver slices for 72 hours with 2-200 microM of either indole-3-carbinol (I3C) or indole-3-acetonitrile (3-ICN) had little effect on cytochrome P-450 (CYP)-dependent enzyme activities, enzyme induction was observed after in vivo administration of I3C. The treatment of rat liver slices with 50 microM 3,3'-diindolylmethane (DIM; a dimer derived from I3C under acidic conditions) for 72 hours resulted in a marked induction of CYP-dependent enzyme activities. DIM appears to be a mixed inducer of CYP in rat liver slices having effects on CYP1A, CYP2B and CYP3A subfamily isoforms. Small increases in liver slice reduced glutathione levels and glutathione S-transferase activity were also observed after DIM treatment. While aflatoxin B1 and monocrotaline produced a concentration-dependent inhibition of protein synthesis in 72-hour-cultured rat liver slices, cytotoxicity was markedly reduced in liver slices cultured with 50 microM DIM. These results demonstrate that cultured rat liver slices may be employed to evaluate the effects of chemicals derived from cruciferous and other vegetables on CYP isoforms. In addition, liver slices can also be utilized to examine the ability of such chemicals to modulate xenobiotic-induced toxicity.     

Metabolic and structural viability of precision-cut rat lung slices in culture

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72?h.

2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with β-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600?µm.

3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8?h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72?h incubation.

4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24?h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24?h; longer incubations did not result in further leakage.

5. It is concluded that precision-cut rat lung slices, of 600?µm thickness, can be maintained metabolically viable in culture for some 8?h.

     

Evaluation of cultured, precision-cut rat liver slices as a model to study drug-induced liver apoptosis

The precision-cut liver slice culture model has been used widely to investigate drug metabolism and drug-induced necrosis. However, apoptosis, a key mediator of liver toxicity remains to be studied in this model. We evaluated apoptosis induced by thioacetamide (TAA) in rat liver slices, and in livers taken from TAA-treated rats as a control. Rat liver slices were treated with 50, 75 and 100 mM of TAA for 15 h. Histopathological examination of the liver slices revealed specific centrilobular localization of apoptotic hepatocytes at 75 mM but randomly distributed at 100 mM. Apoptosis in centrilobular hepatocytes was confirmed by appearance of cleavage products of caspase-3 and DNA fragmentation studied by TUNEL method. A concentration-dependent release of cytochrome c was observed in the slices, suggesting a role for mitochondria in the apoptosis triggered by TAA. The in vitro results were compared to the data obtained in male Sprague-Dawley rats given a single ip injection of 40 mg/kg TAA and sacrificed 1, 2, 3 and 6 h after dosing. Histopathological analyses showed specific centrilobular localization of apoptosis after 6 h treatment. Caspase-3 activation, DNA fragmentation and cytochrome c release were also observed in the liver of rats treated with TAA. Overall these data indicated that precision-cut liver slices provide a valuable in vitro system to study drug-induced liver apoptosis.     

The relationship between 5-hydroxytryptamine and paraquat accumulation into rat lung

The uptake of 5-hydroxytryptamine (5HT) into rat lung slices has been shown to obey saturation kinetics and to be inhibited by imipramine, metabolic inhibitors and a sodium-free medium. The apparent K_m for the uptake process was found to be 3.3 μMwith a V_max of 6 nmoles/g wet wt/min. Lung slices taken from rats given a dose of paraquat known to damage type I and type II lung epithelial cells showed inhibition of paraquat uptake but no inhibition of 5HT uptake. This together with the stimulation of paraquat accumulation into rat lung slices in a sodium-free medium leads to the conclusion that the uptake of paraquat and 5HT into the lung does not occur in the same cell type.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V(max) in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

人肺精确薄片体外移植培养模型的建立及对流感病毒感染的反应

目的建立人肺精确薄片体外移植培养模型;并检测暴露于流感病毒下的人肺薄片里是否存在感染和病毒复制。方法参照改良琼脂糖充填肺组织的方法,以1.5%低熔低胶琼脂糖经充膨、钻取、切片及培养等步骤建立人肺精确薄片体外移植培养模型。选取不同时间点对移植培养的人肺薄片进行显微镜下组织形态观察;以乳酸脱氢酶释放毒性实验评价细胞毒性;以RT-PCR和免疫组织化学方法测定人肺薄片组织对暴露的流感病毒的反应。结果人肺精确薄片模型经长时间(>10d)的培养,维持正常的结构和功能,支持人肺薄片组织暴露于流感病毒后的感染和病毒复制。结论建立的人肺精确薄片体外移植模型可靠和稳定,可被用于流感病毒与人体肺器官感染的研究。     

Acute and subacute inhalation toxicity of diborane in male ICR mice

 To clarify the toxicity of diborane, we conducted acute (15 ppm for 1, 2, 4 or 8 h) and subacute (5 ppm for 2 or 4 weeks) inhalation studies on ICR mice. The concentration resulting in a 50% kill after 4 h exposure was 31.5 ppm. Body weight gain was suppressed and the lung weight was increased in diborane-exposed mice in both acute and subacute studies. In the acute study, diffuse pan bronchiolitis-like lesions developed in the lung in various degrees depending on exposure time, which can be pathologically characterized as infiltration of inflammatory cells into the terminal bronchioles and surrounding alveoli, pulmonary congestion and bleeding and/or edema. In the subacute study, we observed lymphoid hyperplasia in the perivascular and peribronchial areas, and infiltration of macrophage and plasma cells into the alveoli. In the mice exposed for 4 weeks, the lesions were more severe than in those exposed for 2 weeks, consisting of hyperplasia and desquamation of Clara cells. In the nasal cavity, we saw mucous exudate and inflammatory cells, suggesting irritation caused by diborane. The histopathological findings, except for the respiratory organs, did not reveal any exposure-related changes. No significant changes were seen in hematological and serum biochemical examinations either. In conclusion, the target organ of diborane inhalation is the respiratory organs, particularly the lung. Further inhalation experiments are essential to investigate the safety exposure levels of diborane. Received: 31 May 1994 / Accepted: 3 November 1994     

Factors affecting the efflux of paraquat from rat lung slices

In an attempt to reduce the toxicity of paraquat several compounds were examined for their ability to increase the rate of efflux of paraquat from the lung. The compounds were selected because they were known, from in vitro studies, to reduce the accumulation of paraquat into the lung. Histamine (100 μM), promethazine (100 μM), putrescine (100 μM), bromthymol blue (300 μM) and the metabolic inhibitors iodoacetate (1 mM), rotenone (100 μM) and KCN (1 mM) have been shown to reduce the accumulation of paraquat into rat lung slices, as did the incubation of slices under nitrogen.The efflux of paraquat from lung slices prepared from rats dosed intravenously with paraquat was biphasic, having a very fast component and a slow component. The slow component was first order and was characterised by a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 17 h. This half life is similar to that seen in vivo (24h) following intravenous dosing. When lung slices prepared from rats dosed intravenously with paraquat were incubated in the presence of iodoacetate (1 mM) or under nitrogen, the half life of paraquat in the slices was reduced to approximately 3 h. In the presence of rotenone (100 μM) it was reduced to approximately9 h. Histamine (100 μM) and promethazine (100 μM) did not affect the efflux of paraquat from lung slices. Bromthymol blue, a dye which forms “ion pair” complexes with paraquat, also significantly increased the efflux of paraquat from lung slices. The effect of bromthymol blue, however, decreased with time and thus paraquat efflux in the presence of bromthymol blue did not obey first order kinetics.In order to measure cellular viability of lung slices, oxygen comsumption, glucose oxidation and the rate of the efflux of protein from the slices into the incubation medium were determined. Iodoacetate (1 mM) and rotenone (100 μM) almost abolished oxygen consumption and glucose oxidation whereas these activities were inhibited to a lesser extent by bromthymol blue (300 μM) (18% and 30%, respectively). During the first 30 min of incubation in the presence of KCN (1 mM) oxygen consumption was almost abolished but between 30 min and 4 h returned to control levels. The effect of KCN could therefore be divided into 2 phases. Over 4 h incubation glucose oxidation was inhibited by 36%. Iodoacetate (1 mM) and incubation under nitrogen caused the most pronounced increases in the rate of protein efflux from slices. KCN (1 mM) and rotenone (100 μM) also increased the rate of protein efflux but to a lesser extent. We have therefore suggested that the effect of KCN (1 mM) on cellular viability, while severe, may be less than that of iodoacetate (1 mM) or incubation under nitrogen.We have concluded from these studies that: (1) the reduction in the accumulation of paraquat or increase in its efflux produced by metabolic inhibitors may be a consequence of lung cell damage; (2) bromthymol blue and putrescine cause an increase in the efflux of paraquat from the lung in vitro without damaging the tissue suggesting that in principle this approach may be useful in vivo; and (3) histamine and promethazine reduce the uptake of paraquat into the lung by direct competition with paraquat since they neither increase the rate of efflux of paraquat from the lung nor reduce cellular viability.     

Formation of epoxide and quinone protein adducts in B6C3F1 mice treated with naphthalene,sulfate conjugate of 1,4-dihydroxynaphthalene and 1,4-naphthoquinone

 Naphthalene (NA) is metabolically activated to the reactive intermediates, naphthalene oxide (NO) and naphthoquinones. To investigate the role of circulating reactive metabolites in NA toxicity, the half-life of NO was examined. The in vitro half-life of NO in both whole blood and plasma was 10 min. Detectable levels of NO were seen in perfusate leaving the isolated perfused liver of B6C3F₁ mice infused with 10 μmol/h NA. Identification of protein sulfhydryl adducts in NA-exposed mice (50 and 100 mg/kg, IP, 24 h) revealed a predominance of quinone adducts in liver, lung, kidney, red blood cells and brain. The epoxide adduct predominated in plasma protein. Administration of the sulfate conjugate of 1,4-dihydroxynaphthalene (NHQS) (100  mg/kg) resulted in formation of naphthoquinone protein sulfhydryl adducts in lung, liver and kidney. Administration of 1,4-naphthoquinone (NQ) (5 mg/kg) produced NQ adducts in liver, lung, kidney, plasma and brain. Received: 5 August 1994 / Accepted: 19 October 1994