Effect of carbon tetrachloride treatment on ethanol metabolism

Administration of a single dose (2.5 ml/kg body weight, p.o.) of carbon tetrachloride to rats was found to cause a marked decrease in activity of the hepatic microsomal ethanol oxidizing system (MEOS). As early as 6 hr after CCl₄ administration 50 per cent decrease of MEOS activity was observed; this decrease amounted to 58 and 63 per cent at 10 and 20 hr respectively. With identical CCl₄ treatment, there was no change in hepatic alcohol dehydrogenase activity. At 20 hr, when the reduction of MEOS was greatest, there was no significant effect of the CCl₄ on the rate of ethanol uptake by liver slices or on the rate of ethanol metabolism in vivo as measured in the whole body or as estimated from the rate of decrease of blood ethanol concentration. It is, therefore, suggested that MEOS does not play a significant role in ethanol metabolism in vivo in the rat.     

Spermatocyte toxicity of 2-methoxyethanol in vivo and in vitro: Requirement for an intact seminiferous tubule structure for germ cell degeneration

Cultures of isolated testicular cells are widely used for evaluating mechanisms of action of direct-acting testicular toxicants. However, for testicular cells, the expression of toxicity in vitro is frequently different from that found in vivo. 2-Methoxyethanol (ME) produces testicular lesions in rats which are characterized by pachytene spermatocyte degeneration 24 hr after dosing. As part of a study to evaluate mechanisms of male germ cell toxicants, we compared the morphological aspects of spermatocyte toxicity after in vivo exposure to ME with those found in various testicular cell culture systems after in vitro exposure to the active ME metabolite, 2-methoxyacetic acid (MAA). Immature rats were used because they respond to in vivo ME treatment in the same way as adults, but their testes are relatively enriched in pachytene spermatocytes. 24-day-old rats were given a single dose of 250 mg ME/kg body weight by gavage and the testes were evaluated 24 hr after dosing. In vitro, cultured seminiferous tubules, Sertoli-germ cell co-cultures, and enriched mixed germ cells, all prepared from 24-day-old rats, were evaluated after 24-hr in vitro exposure to 5 m MAA (a level of MAA found after ME exposure in vivo). Testes from ME-exposed rats showed the expected pachytene spermatocyte degeneration 24 hr after dosing. Similar changes were observed in cultured seminiferous tubules after 24 hr of exposure to MAA in vitro. However, without the intact seminiferous tubule structure in vitro, the expression of MAA spermatocyte toxicity was different. In conventional Sertoli-germ cell co-cultures, spermatocyte detachment from the Sertoli cell monolayer occurred as expected, although no significant morphological degeneration was observed in these detached germ cells. Similarly, no increase in degenerating spermatocytes was noted in isolated enriched mixed germ cells after in vitro MAA exposure. Instead, toxicity in these germ cell fractions was expressed only by increased uptake of trypan blue dye, revealing an increase in plasma membrane permeability. In summary, this in vivo/in vitro comparison of the spermatocyte toxicity of ME/MAA showed that the expression of toxicity is different in the different culture architectures and that an intact seminiferous tubule structure is required for full expression of the morphological degeneration produced by ME/MAA, and suggests the usefulness of culture seminiferous tubules in future mechanistic studies.     

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

Thyroxine (T₄)-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague–Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T₄-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T₄-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), β-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T₄-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T₄-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T₄-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T₄-UGT activities.     

Prediction of drug-drug interaction from In vitro plasma protein binding and metabolism: A study of tolbutamide-sulfonamide interaction in rats

The prediction of tolbutamide-sulfonamide interaction from in vitro unbound intrinsic clearance was studied by comparing the in vvivo and in vitro total body clearance (CL_tot) in rats. The The sulfonamides used were sulfaphenazole (SP), sulfadimethoxine (SDM) and sulfamethoxazole (SMZ). The plasma half-life (T_1/2) of tolbutamine (TB) was increased ssignificantly by SP and SDM, while CL_tot was decreased significantly by both drugs; no significant changes were observed in their parameter by SMZ. The in vitro plasma protein binding and microsomal oxidation of TB were competitively inhibited by sulfonamides; the order of inhibitor constantswas SMZ>SP>SDM. Since metabolism is the rate-determining step of the hepatic extraction of TB, the in vivo CL_tot can be expressed by the equation; CL_tot ƒ_BCL_int, where ƒ_B is the blood free fraction and CL_int is the hepatic intrinsic clearance of unbound drug. A comparatively good agreement was observed between the in vivo CL_tot and that calculated from both in vitro plasma protein binding and metabolic parameters.     

Liposomal polyethyleneglycol and polyethyleneglycol-peptide combinations for active targeting to liver in vivo

This report describes the development and evaluation of a range of polyethyleneglycol and polyethyleneglycol-peptide liposome formulations that effectively target liver in vivo. A 19-amino-acid sequence from the N-terminal region of the circumsporozoite protein of Plasmodium berghei was attached to the distal end of di22:1-aminopropane-polyethyleneglycol₃₄₀₀, and incorporated into liposomes containing di22:1-phosphatidylcholine and di22:1-phosphatidylethanolamine-polyethyleneglycol₅₀₀₀. By systematically varying the mole fractions of both the lipid-polyethyleneglycol and the lipid-polyethyleneglycol-peptide conjugates, and screening for serum-induced aggregation in vitro, a serum-stable range of formulations was established. These stable formulations were tested for binding to Hepa 1-6 liver cells in culture, and from these results three formulations were prepared for intravenous administration in mice. All three formulations exhibited effective liposome targeting to the liver, with approximately 80% of the total injected dose recovered in the liver within 15 min. Uptake by liver cells was more than 600-fold higher than uptake by those in the heart, and more than 200-fold higher than uptake by lung or kidney cells. Effective targeting to liver in vivo was successful after repeated (up to three) administrations to the host at 14-day intervals. All formulations prepared for in vivo administration were stable in the presence of serum, as measured by complete retention of entrapped calcein dye. The formulation with the lowest mole fractions of peptide and polyethyleneglycol was the most cost-effective in terms of encapsulation efficiency and minimal use of peptide and polymer compounds. The in vitro biophysical screening, followed by cell culture testing, reduced the number of animals required to develop an effective set of targeted liposome formulations for in vivo application.     

In vitro dermal absorption of pesticides: IV. In vivo and in vitro comparison with the organophosphorus insecticide diazinon in rat, guinea pig, pig, human and tissue-cultured skin

In vitro skin absorption tests are currently being developed as an alternative to in vivo animal tests for predicting the degree of occupational exposure to pesticides. In the study reported here, in vitro percutaneous absorption tests were conducted with the ¹⁴C-ring-labelled pesticide, diazinon, dissolved in acetone and applied to the dermatomed skin (0.5 mm) of a number of species at a dose rate of 9.5–16.7 μg/cm². Skin permeation was determined for 48 hr after exposure using an in vitro flow-through system. Skin permeation was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution and the percentage recovery obtained in methanol washes of the skin at 48 hr and in skin digests. Listed in decreasing order, the total percentage in vitro dermal absorptions (mean ± SD) obtained by 48 hr after exposure for the five skin types were: 47 ± 3.4% (rat), 36 ± 0.9% (tissue cultured Testskin), 33 ± 2.8% (hairless guinea pig), 20 ± 3.1% (human) and 15 ± 13.1% (pig). The percentage recoveries in soapy water skin washes at 24 hr, in methanol washes and skin digests at 48 hr and of ¹⁴C-labelled volatiles collected in air traps at 48 hr after exposure are reported. Comparative in vivo studies demonstrated 37 ± 0.8 and 24 ± 5.7% recovery of ¹⁴C in the urine of rats (dose rate, 6 μg/cm²) and hairless guinea pigs (dose rate, 5 μg/cm²), respectively, by 14 days after exposure. Total faecal recovery 14 days after exposure was 18 ± 0.4 and 4 ± 0.9% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after exposure demonstrated a total tissue recovery of 0.6 ± 0.1% [¹⁴C]diazinon in rats and 1 ± 0.2% in hairless guinea pigs. The total recovery in skin removed from the dose site at 14 days after exposure was 0.2 ± 0.02% and 0.1 ± 0.05% in rats and hairless guinea pigs, respectively. Recovery of radioactivity from soapy water skin washes conducted at 24 hr after exposure was 21 ± 3.8% for rats and 2 ± 0.1% for hairless guinea pigs. Recovery in skin patches was 23 ± 5.4% and 73 ± 2.9% in rats and hairless guinea pigs, respectively. The in vitro data for dermal absorption of [¹⁴C]diazinon for rats (47 ± 3.4%) and hairless guinea pigs (33 ± 2.8%) were in good agreement with the data observed for rats (56 ± 1.03%) and hairless guinea pigs (28 ± 6.0%) in vivo. This study supported the use of in vitro skin absorption tests as an alternative to in vivo animal testing.     

Hormonal regulation of superoxide dismutase, catalase, and glutathione peroxidase activities in rat macrophages

This study examined the effects of glycocorticoids, insulin, thyroxine, and epinephrine upon the activities of CuZn- and Mn-superoxide dismutases (SOD), catalase, and glutathione peroxidase (GPX) and upon hydrogen peroxide production in rat macrophages obtained from the intraperitoneal cavity. The experiments were performed in vivo under conditions causing hormonal dysfunctions: adrenal demedullation, dexamethasone treatment, thyroidectomy, administration of -tri-iodothyronine (T₃) and -thyroxine (T₄), and diabetes. Macrophages were also cultured for 24 hr in the presence of dexamethasone, thyroid hormones, and insulin as to evaluate possible interferences caused in vivo by changes in other hormones. The results indicated that these hormones do control the activities of the antioxidant enzymes and hydrogen peroxide production both in vivo and in vitro. Insulin increased the activities of CuZn-SOD, catalase, and GPX and reduced that of Mn-SOD. Thyroid hormones raised the activities of CuZn- and Mn-SOD and decreased that of GPX, whereas glucocorticoids reduced both Mn-SOD and GPX. The removal of the adrenal medulla caused a decrease of Mn-SOD and GPX activities in the macrophages. Hydrogen peroxide production was increased by insulin and reduced by thyroid hormones and glucocorticoids. The changes in antioxidant enzyme activities caused by these hormones in macrophages may indicate important mechanisms for the establishment of impaired immune function in endocrine pathologies.     

In vitro dermal absorption of pesticides: VI. In vivo and in vitro comparison of the organochlorine insecticide DDT in rat, guinea pig, pig, human and tissue-cultured skin

In vitro dermal absorption tests were conducted with the ¹⁴C-ring-labelled pesticide, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 16–27 μg/cm². Skin absorption was determined for 48 hr after exposure using in vitro flow-through cells. Skin absorption was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution added to the percentage recovery for the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline (used with Moody aluminium cells), and Hanks' HEPES buffered saline with 4% serum albumin (used with Bronaugh flow-through cells) were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 42 ± 2.6% [hairless guinea pig; Hanks' receiver (HR)], 34 ± 10.5% (rat; HR), 28 ± 13.2% [Testskin; Ringer's receiver (RR)], 28 ± 2.9% (human; HR), 22 ± 3.3% (Testskin; HR), 18 ± 6.2% (pig; RR) and 14 ± 2.1% (pig; HR). The percentage ¹⁴C activity recovered in soapy water rinses of the skin specimens at 24 hr, and for methanol skin washes and skin digests at 48 hr, and of ¹⁴C-labelled volatiles collected in air traps are reported. Data obtained with pig and Testskin for DDT using the Moody flow-through permeation cell was compared with that obtained using the Bronaugh cell. Significantly greater (P < 0.05) percentage recovery was obtained for the soap washes at 24 hr of the skin following the Bronaugh procedure than was obtained with the Moody method. Comparative in vivo studies demonstrated urinary recovery was 2 ± 0.5 and 15 ± 1.7% for rats (dose rate; 6 μg/cm²) and guinea pigs (dose rate: 9 μg/cm²), respectively. Total faecal recovery was 20 ± 1.9 and 44 ± 2.75% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after dosing demonstrated total tissue recovery of 51 ± 5.6% in rats but of only 3 ± 0.7% in guinea pigs. Including the ¹⁴C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 73 ± 5.9 and 62 ± 4.1% for rats and guinea pigs, respectively. Recovery of ¹⁴C from soapy water skin washes conducted at 24 hr after exposure was 3 ± 1.4 and 14 ± 1.8% for rats and guinea pigs, respectively, and this was significantly less than that obtained by both the Bronaugh and Moody in vitro procedures. Skin patch recovery was 24% for both rats and guinea pigs. In summary, the in vitro data underestimed the degree of dermal absorption observed in vivo for both rats and guinea pigs, and this was thought to be due to an overly vigorous removal of the pesticide skin deposit by the soap washing procedures used in vitro in comparison with the in vivo washing procedure.     

In vitro dermal absorption of pesticides: V. In vivo and in vitro comparison of the herbicide 2,4-dichlorophenoxyacetic acid in rat, guinea pig, pig, human and tissue-cultured skin

In vitro dermal absorption tests were conducted with the ¹⁴C-ring-labelled herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 7–8 μg/cm². Skin absorption was determined for 48 hr after exposure using an in vitro flow-through system. Skin absorption was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution and the percentage recovery in the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline, and Hanks' HEPES buffered saline with 4% serum albumin were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 47 ± 4.3% [tissue cultured Testskin; Hanks' receiver (HR)], 40 ± 4.5% (rat; HR), 19 ± 1.8% (human; HR), 14 ± 2.3% (hairless guinea pig; HR), 14 ± 8.8% (pig; Ringer's receiver). The percentage recovery of the radiolabel in soapy water skin washes at 24 hr, methanol washes and skin digests at 48 hr, and of ¹⁴C-labelled volatiles collected in air traps at 48 hr after exposure are reported. Comparative in vivo studies were conducted for 14 days after exposure and demonstrated 32 ± 3.9 and 28 ± 7.8% recovery of ¹⁴C in the urine of rats (dose rate, 3 μg/cm²) and guinea pigs (dose rate, 4 μg/cm²), respectively. Total faecal recovery was 2 ± 0.3 and 9 ± 3.5% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 day after dosing demonstrated a total tissue recovery of ¹⁴C activity of 1 ± 0.1 and 2 ± 0.5% in rats and guinea pigs, respectively. Including the ¹⁴C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 49 ± 10.4 and 40 ± 9.9% in rats and guinea pigs, respectively. Recovery of ¹⁴C activity from soapy water skin washes conducted at 24 hr after exposure was 28 ± 8.1 and 43 ± 9.0% for rats and guinea pigs, respectively. Recovery in skin patches was 18% (guinea pigs) and 26% (rats). In summary, the in vitro/in vivo concordance for the rat dermal absorption data was good but the in vitro data for hairless guinea pigs underestimated the in vivo absorption, and therefore for 2,4-D, rat skin may provide a better model of percutaneous absorption.     

Evaluation of the cytotoxicity of 10 chemicals in human and rat hepatocytes and in cell lines: Correlation between in vitro data and human lethal concentration

The cytotoxicity of 10 chemicals from the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) list (nos 21–30) was evaluated in human and rat cultured hepatocytes and in two established cell lines (HepG2 and 3T3) according to the MEIC programme organized by the Scandinavian Society of Cell Toxicology. The MTT test was used as the endpoint of cytotoxicity after 24hr of exposure to the chemicals. Theophylline, phenobarbital and paraquat were the least cytotoxic compounds in the cellular systems (IC₅₀ = 450-17,000 μm) except for the 3T3 cells. The seven remaining chemicals (dextropropoxyphene, propranolol, arsenic trioxide, cupric sulfate, mercuric chloride, thioridazine and thallium sulfate) showed a similar relative cytotoxic ranking in the four in vitro systems in the lower range of concentrations (IC₅₀ = 2–350 μm). The data suggest that these 10 chemicals have a basal cytotoxic effect common to the four in vitro systems, and probably none of these compounds could be considered either hepatotoxic or species specific. The correlation between in vitro data and human lethal blood concentrations showed that the predictability of the in vitro systems was similar to that of in vivo rodent tests (LD₅₀) only when low cytotoxic concentrations (IC₁₀) were used for correlation.     

A new technique for culturing rat embryos between gestation days 14 and 15

A method for culturing rat embryos between days 14 and 15 has been developed. On gestation day 14 (morning of sperm and PLUG = day 0), laparotomies were performed on time-mated rats that had been anaesthetized with halothane. The embryos, with open attached yolk sacs and placentas, were cultured in 650-ml plastic flasks with 120 ml medium (0.7% bovine serum albumin in Earle's balanced salt solution with antibiotics, prewarmed to 37°C and continuously flushed with water-saturated 95% O₂/5% CO₂, pH 7.4) in a Dubnoff metabolic shaker with gentle agitation. Viability and morphology were assessed after 26 hr in culture. On day 15, embryonic growth and development were similar to that in in vivo controls, and viability was high (55%).     

In vitro cytotoxicity tests for the prediction of acute toxicity in vivo

Investigations of the use of in vitro cytotoxicity tests for the prediction of acute toxicity in vivo have been reviewed with particular emphasis on those studies that have been published during the past 5 years. Numerous cell types, endpoints and exposure periods have been used in cytotoxicity tests, although these appear generally to have little effect on the resulting correlation between in vitro IC₅₀ values and in vivo LD₅₀ values. The in vitro data correlate better with rodent parenteral (ip or iv) LD₅₀ values than with oral LD₅₀ values due to kinetic considerations. For certain groups of related chemicals (e.g. antitumour compounds, metal salts), and for some sets of unrelated chemicals, the in vitro data correlate very well with LD₅₀ values. However, while cytotoxicity tests are useful for screening chemicals for their intrinsic and relative toxicities, it is impossible to tell whether predictions based on cytotoxicity data alone would be sufficiently accurate for labelling and classifying a new chemical according to its likely acute toxicity in vivo. The in vitro endpoints need to be of greater relevance to the possible mechanisms of chemically-induced acute toxicity in vivo than most of those that are used at present.     

Eye irritation: Reference chemicals data bank

A list of 55 chemicals has been developed for which comprehensive in vivo rabbit eye irritation data are available. No new in vivo testing has been carried out to qualify a chemical for inclusion in the list. The 55 chemicals selected are available at high and consistent purity and are expected to be stable on storage. They have been tested undiluted in in vivo studies, except those chemicals where high concentrations of the substance could be expected to cause severe effects. The in vivo data have been generated since 1981 in studies carried out according to OECD Test Guideline 405 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three rabbits evaluated at the same time, involving instillation of 0.1 ml (or equivalent weight) into the conjunctival sac, and in which observations were made at least at 24, 48 and 72 hr after instillation. The chemicals represent a range of chemical classes (acetates, acids, alcohols, alkalis, aromatics, hydrocarbons, inorganics and surfactants) and different degrees of irritancy. They are ranked for eye irritation potential on the basis of a ‘modified maximum average score’. The reference data bank should be of use in validation tests for promising alternatives to the in vivo rabbit eye irritation test. This is an essential step in the progression to regulatory acceptance of alternative procedures.     

Cadmium-induced metallothionein synthesis in the rat liver slice system

In vivo exposure of a rat to cadmium results in elevation of the hepatic metal-binding protein, metallothionein (MT). The present work describes the induction of MT in the in vitro liver slice system. Incubation of rat liver slices with CdCl₂ resulted in a dose-dependent elevation of tissue MT. The cadmium-binding protein was increased by 140 and 220% in the presence of 10 and 20 μ CdCl₂, respectively. A lower level (5 μ ) of the inducer had only a slight effect. A time-course study showed a gradual increase in the MT level following incubation of the liver slices for 4 and 6 hr with 10 μ -cadmium. Characterization of the metal-binding protein by Sephadex G-75 gel filtration revealed that it is composed of MT and also of a high-molecular-weight fraction that might be either a polymerized or aggregated form of MT, or another type of cadmium-binding protein. These findings indicate that the response of the liver slice system to toxic agents is similar to that of the intact animal. These are encouraging results which need to be extended before the system can be introduced into the routine screening of hepatotoxic agents.     

Nanoparticulate delivery system for insulin: Design, characterization and in vitro/in vivo bioactivity

Insulin-loaded alginate–dextran nanospheres were prepared by nanoemulsion dispersion followed by triggered in situ gelation. Nanospheres were characterized for mean size and distribution by laser diffraction spectroscopy and for shape by transmission electron microscopy. Insulin encapsulation efficiency and in vitro release were determined by Bradford protein assay and bioactivity determined in vitro using a newly developed Western blot immunoassay and in vivo using Wistar diabetic rats. Nanospheres ranged from 267 nm to 2.76 μm in diameter and demonstrated a unimodal size distribution. Insulin encapsulation efficiency was 82.5%. Alginate–dextran particles suppressed insulin release in acidic media and promoted a sustained release at near neutral conditions. Nanoencapsulated insulin was bioactive, demonstrated through both in vivo and in vitro bioassays     

Role of lipid peroxidation in the toxicity of T-2 toxin

A. , G. , B. and W. . Role of lipid peroxidation in the toxicity of T-2 toxin. Toxicon 25, 1321 – 1328, 1987.—Recent reports suggest that lipid peroxidation may be involved in the toxicity of T-2 toxin. In the present study the influence of T-2 toxin on two parameters of lipid peroxidation was examined: the formation of thiobarbituric acid reactive material in isolated hepatocytes and liver homogenates from rats and ethane exhalation in vivo. In isolated hepatocytes there was no significant increase in thiobarbituric acid reactive material, neither after addition of T-2 toxin in vitro nor when the toxin had been applied to the rats 15 hr before preparation of hepatocytes. In liver homogenates the amount of thiobarbituric acid reactive material was increased up to 50% over the controls, depending on the dose of T-2 toxin. The increased values are difficult to interpret, because the extent of the increase depends on the method used for determination of thiobarbituric acid reactive material. Measuring another parameter of lipid peroxidation, i.e. ethane exhalation, there was no difference between the T-2 toxin treated rats and the controls whereas carbon tetrachloride treated rats exhaled high amounts of ethane. These results suggest that lipid peroxidation does not play a major role in T-2 toxin toxicity.     

Determination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements

Biologically based scaling factors have to be used to predict in vivo metabolic clearance of xenobiotics from data obtained in vitro. Although standard values for the hepatocellularity numbers for different species are used in the literature, detailed information on the determination of these values has only been presented for humans and rats, and somewhat different results have been obtained in different studies. The present work was undertaken in order to determine the number of hepatocytes per gram of liver for human, dog, rabbit, rat and mouse livers. Hepatocellularity numbers were calculated from the ratio between the liver protein concentration and the protein concentration in the corresponding hepatocyte suspension. For human, rabbit, rat and mouse livers, the hepatocellular values were in the same range, more precisely 139 ± 25, 114 ± 20, 117 ± 30 and 135 ± 10 million cells per gram of liver, respectively. However, for the dog liver, the corresponding value was as high as 215 ± 45 million cells per gram. These values should be of importance during the scaling process of intrinsic clearance for xenobiotics in hepatocytes to in vivo hepatic clearance.     

Comparison of cytotoxic effects of chemicals in four different cell types

Cytotoxic effects were compared using a colony-formation assay in three established cell lines (Balb 3T3, mouse whole embryo, ARLJ301-3, rat liver and FRSK, rat keratinocytes) and one primary cell culture (RC-1, rabbit cornea) with the Draize eye irritancy score in vivo. The cells were treated with 52 chemicals on the day after plating, then cultured for 7 or 8 days. The 50% inhibition dose (ID₅₀) for each chemical was calculated based on the colony number. With a few exceptions, the cytotoxicities of the chemicals were in the following order in all four cells: cationic detergents anionic detergents non-ionic detergents glycol or oil. These results were almost the same as the data in vivo. The correlation coefficients of the ID₅₀ to the Draize score of 20 in vivo were 0.57 (Balb 3T3), 0.61 (ARLJ301-3), 0.71 (FRSK) and 0.65 (RC-1). Balb 3T3 and ARLJ301-3 cells were slightly more sensitive to chemicals than FRSK and RC-1 cells. These results suggest that the colony-formation assay using established cell lines is an attractive method for the screening of chemicals in that large differences among cell types in their response to direct-acting chemicals, were not observed.     

复方麝香黄芪滴丸的体外及体内血清化学成分分析

目的:应用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱(UHPLC-Q-Orbitrap HRMS)对复方麝香黄芪滴丸的体外及体内血清化学成分进行快速分析鉴别.方法:采用Accucore C18(100 mm×2.1 mm,2.6 μm)色谱柱,以0.1％甲酸水溶液-甲醇梯度洗脱,流速0.2mL·min-1,进样量5...