Application of the post-implantation rat embryo culture system to in vitro teratogenicity testing

Originally developed for studying basic mechanisms in developmental biology, the post-implantation embryo culture system has been extended and applied to the testing of chemicals in the field of prenatal toxicology. The endpoints used in a procedure that has been developed for the routine short-term assessment of teratogenicity in vitro are growth (crown-rump length), differentiation (somite number) and morphology (together with measurement of yolk-sac growth and vascularization) in rat embryos explanted on day 9.5 of gestation and cultured for 48 hr in 100% homologous rat serum containing the test compound. Studies involving exposure to trichlorophenoxyacetic acid and to acetylsalicylic acid demonstrate the ability of this system to distinguish between non-specific toxicity and specific effects on development.     

The teratogenic potential of alkoxy acids in post-implantation rat embryo culture: structure-activity relationships

Alkoxy acids are the active metabolites of teratogenic glycol ethers. To examine the relationship of chemical structure to embryotoxicity, the effects of 6 acids on the development of 9.5-day rat embryos over 48 h in culture were studied. Methoxyacetic acid (MAA) and ethoxyacetic acid (EAA) (5 mM) were growth-retarding and induced gross structural defects, with MAA being more effective. n-Propoxyacetic acid (n-PAA) and n-butoxyacetic acid (n-BAA) (5 mM) were markedly less embryotoxic and produced only minor anomalies. Thus, the activities of these substituted acetic acids decreased with the increase in the length of the alkoxy chain. 3-Methoxypropionic acid (3-MPA) and 4-methoxybutyric acid (4-MBA) (5 mM) were much less active than MAA and induced only minor defects. Therefore in this series: RO(CH2)nCOOH, an increase in the value of n caused a greater reduction in embryotoxicity than did an increase in chain length of the alkyl group R.     

利用小鼠全胚胎培养研究环磷酰胺致畸作用

本文应用小鼠全胚胎培养技术 ,通过妊娠 d8分别 ip5,1 0 ,1 5和 2 0 mg·kg-1环磷酰胺 ( CP) ,研究了该药对器官原基形成期胚胎的致畸作用 ,并对其致畸机理作了初步探索 .给药 4h后取胚胎进行培养 ,于 d 1 0 .5收获胚胎 ,测量其卵黄囊直径 ,头长及颅臀长并记录其大体形态的变化 .结果表明 ,1 5mg· kg-1组尾畸形率最高 ;2 0 mg· kg-1组生长迟缓率最高 .电镜观察所显示的细胞凋亡的形态学变化及 DNA琼脂糖凝胶电泳的结果均提示 CP致畸作用可能与其诱导的细胞凋亡有关 .     

Assessment of the teratogenic potential of acrolein and cyclophosphamide in a rat embryo culture system

Rat conceptuses were explanted from the uterus on day 10.5 of gestation. The embryos within the yolk-sacs were then transferred to culture bottles containing pure male rat serum either with or without liver microsomes and NADPH. Acrolein (AC), present worldwide in the environment and one of the intermediate metabolites of cyclophosphamide (CPA), was added to these culture mediums. The conceptuses were grown for a period of 48 h after which the morphological features and their degree of differentiation were examined and the DNA and protein contents determined. The effects produced by AC were compared with those obtained by CPA treatment, using the same culture conditions. AC treated embryos and yolk-sacs showed slight but statistically significant inhibition of growth at concentrations of 100 μM and 150 μM. Higher dose levels (200 μM and 250 μM) resulted in a drastic inhibition of growth and differentiation. However, no gross structural defects were observed at the dose-levels used. In contrast, conceptuses cultivated in the presence of CPA (350 μM), liver microsomes and NADPH showed characteristic morphologic lesions. Our findings indicate that AC is lethal to embryos within a narrow dose-range, but has no teratogenic potential. Therefore, AC is not the metabolite which is responsible for the teratogenic effects observed after CPA treatment in vivo. The results also demonstrate that the postimplantation embryo culture system can discriminate between embryolethal and teratogenic effects and that whole embryos in culture can respond to teratogens in a manner similar to embryos exposed in vivo.     

The metabolism of cyclophosphamide by isolated rat hepatocytes

The metabolism of cyclophosphamide was studied in vitro using isolated rat hepatocytes and mass spectrometry. The major product of primary oxidative metabolism in hepatocytes from phenobarbital treated rats was 4-hydroxycyclophosphamide, isolated as the O-ethyl derivatives, but dechloroethylation was also a substantial pathway. 4-Hydroxycyclophosphamide was converted mainly into carboxy phosphamide and the formation of 4-ketocyclophosphamide was a minor pathway. Evidence is presented that under certain conditions a substantial amount of an O-glucuronide of 4-hydroxycyclophosphamide was formed. The pattern of metabolism in hepatocytes otherwise resembled qualitatively that observed previously in vitro using subcellular fractions and in vivo, but quantitative differences were found. The metabolism of cyclophosphamide by hepatocytes resembles more closely that in vivo than does the metabolism in subcellular fractions, and hepatocytes should be the preferred in vitro system for studying the metabolism of anti-tumour agents.     

Biotransformation of trichloroethylene in collagen gel sandwich cultures of rat hepatocytes

The collagen gel sandwich culture of hepatocytes has been proposed as one of the most suitable culture models available for biotransformation studies of xenobiotics. It is a complex model which imitates the cascade of enzymatic events of in vivo biotransformation and allows investigation of biological endpoints under realistic conditions. The biotransformation of trichloroethylene (TRI) has been studied in this model using rat hepatocytes. Headspace gas chromatographic measurements revealed that hepatocytes, cultured for 4 days in this in vitro system, metabolised TRI into the major oxidative metabolites trichloroacetic acid (TCA) and trichloroethanol (TCE). Cultured hepatocytes were exposed either to TRI, or to TCA and TCE. Endpoints studied were albumin secretion and the cytochrome P450 (CYP)-dependent enzymatic activities ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD) and N-nitrosodimethylamine demethylase (NDMA). The results show that both the parent compound and its metabolites exert specific effects on different CYP-dependent mono-oxygenase activities, as seen in vivo. It is suggested that collagen gel sandwich cultures represent a useful in vitro model for the investigation of metabolism-linked toxicity studies.     

Effects of carbon tetrachloride on embryonic development studied in the post-implantation rat embryo culture system and in chick embryos in ovo

The effects of carbon tetrachloride on embryonic development were investigated in a mammalian and a non-mammalian system. In the former, whole-rat embryos, taken at 9.5 days of gestation, were exposed in vitro to different concentrations of CCl₄ (10, 100, 300, 600 and 1000 μg/ml) in rat serum with or without a rat liver microsomal activating system (S-9 mix). In the latter system, chick embryos in ovo were exposed to different concentrations of CCl₄ vapour (25, 35 and 75 ppm). When studied in the whole-rat embryo culture system without metabolic activation, concentrations of up to 300 μg CCl₄/ml had no effect on the overall development. Concentrations -600 μg CCl₄/ml affected the somite number, growth and morphology of the embryos, which can be interpreted as general toxicity. In the presence of S-9 mix, toxicity occurred at concentrations -300 μg/ml. In ovo exposure to CCl₄ showed that 25 ppm caused a decrease in the number of somites. At 35 ppm, CCl₄ induced further toxicity, as indicated by reduced somite number and growth and increased malformation rates. The results indicate that effects on morphogenic events appeared in both systems at concentration levels that also affected the overall development and that, independently of the choice of species or route of administration, CCl₄ has no potential to induce specific malformation patterns. The presence of a metabolic system in the rat embryo cultures approximately doubled the toxicity of CCl₄.     

环磷酰胺对大鼠早期胚胎发育里程的影响

目的：为探索烷化剂对哺乳动物早期胚胎发育里程的影响及其可能的机制。方法：孕大鼠第３ 天ｉｐ 环磷酰胺（Ｃｙｃ １０ 、２０ 、４０ｍｇ／ｋｇ） ,孕第４ 天评价胚胎的胚胎化作用,胚胎发育时相,及蜕变胚胎率;同时显微外科分离胚胎内细胞团及滋养层细胞,评价两群细胞受损情况。结果：在Ｃｙｃ １０ｍｇ／ｋｇ 时,胚胎的胚泡化作用,胚胎发育时相均未受明显影响;４０ｍｇ／ｋｇ 时,蜕变胚胎率明显提高。胚泡化率及胚胎细胞数呈剂量依赖性减少,其中内细胞团细胞减少尤甚。结论：Ｃｙｃ 在未引起早期胚胎蜕变率显著增加时,可影响细胞周期,但不影响发育里程;高剂量时,显著影响发育里程并伴蜕变率显著增加     

Teratogenicity of cyclophosphamide in a coupled microsomal activating/embryo culture system

Using the coupled microsomal activating/embryo culture system, in vitro experiments were performed to establish the role of metabolism in the embryo toxicity and teratogenicity of cyclophosphamide. Cyclophosphamide in the coupled microsomal activating/embryo culture system produced characteristic morphological lesions as well as a general inhibition of embryo and yolk sac growth. Increasing concentrations of NADPH in the presence of microsomes and cyclophosphamide produced progressively greater responses. These effects did not occur when microsomes and NADPH were present in the serum medium for the first 2 hours of incubation followed by one washing and then culturing of the conceptuses from hr 2 to hr 48 in a medium containing cyclophosphamide alone. Cytochrome P-450-depleted microsomes did not bioactivate cyclophosphamide to teratogenic or toxic metabolites. The results indicate that cytochrome P-450-dependent microsomal metabolism of cyclophosphamide is required for the embryotoxic and teratogenic effects observed in vitro.     

Transfection of adult primary rat hepatocytes in culture

The use of adult primary hepatocytes in culture is of importance for the understanding of hepatic processes at the cellular and molecular levels, and the possibility to employ transient transfection of reporter constructs is invaluable for mechanistic studies on hepatic gene regulation. Although frequently used, there is a lack of reports addressing optimization and characterization of transfection of primary rodent hepatocytes. Here, we have shown that the efficiency of biochemical transfection reagents varies significantly and that Lipofectamine2000 was a superior transfection reagent for adult primary rat hepatocytes when using luciferase reporter vectors. The efficiency increased when the cells were allowed ample time to adapt to the in vitro milieu. Cotransfection of a second reporter gene indicated a risk for promoter competition, and we found that relating reporter activity to total cellular protein content gave consistent and reliable results. Differentiation of the cells, achieved by including biomatrix from the Engelbreth-Holm-Swarm mouse sarcoma in the culture system, was to a larger extent required for hormonal/drug responses of transfected constructs than for responses of endogenous genes and assured responses of transfected constructs. Dexamethasone (Dex) is most often included in hepatocyte culture media, but we could not demonstrate a general beneficial effect of Dex on expression of luciferease reporter contructs. Using the established protocol, we have demonstrated responses of transfected constructs to growth hormone, glucocorticoid and LXR stimuli.     

牛磺酸对原代培养大鼠肝细胞的毒性作用

目的用原代培养正常大鼠肝细胞研究牛磺酸(β氨基酸)对肝细胞的毒性作用。方法用2步灌注法分离原代大鼠肝细胞;用MTT法测定细胞活力并计算IC50,观察药物作用后,培养液上清中AST、ALT和LDH活性及培养液中GSH和细胞内GSH的含量。结果细胞生长抑制率与剂量成正相关性,牛磺酸的IC50为24.23g.L-1。高浓度牛磺酸组培养液上清中AST、ALT和LDH活性显著升高,GSH含量显著减少。结论高浓度的牛磺酸对体外培养的肝细胞有一定损伤。     

Isoniazid-induced hepatotoxicity in rat hepatocytes of gel entrapment culture

Gel entrapment culture of rat hepatocytes in hollow fibers were evaluated as a potential in vitro model for studies on isoniazid-induced hepatotoxicity. After exposure to isoniazid (0.11 mM and 1.1 mM) for 24-96 h, gel entrapped hepatocytes were more severely damaged than hepatocyte monolayers according to the assays on methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, and albumin secretion. Furthermore, CYP 2E1 activity detected by 4-nitrocatechol (4-NC) formation maintained at least 7 days in gel entrapped hepatocytes but decreased to an undetectable level within 2 days in hepatocyte monolayer. And the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), significantly reduced isoniazid-induced GSH depletion in gel entrapped hepatocytes. In addition, the protective effects of N-acetylcysteine (NAC), GSH, liquorice extract and glycyrrhizic acid (GA), a purified compound from liquorice extract, against isoniazid hepatotoxicity were clearly observed in gel entrapped hepatocytes at 72 h incubation. Overall, gel entrapped hepatocytes were more susceptible to isoniazid-induced hepatotoxicity than hepatocyte monolayers by a possible mechanism that higher CYP 2E1 activity in gel entrapped hepatocytes could enhance isoniazid toxicity. This indicates that gel entrapped hepatocytes in hollow fibers could be a more effective model than hepatocyte monolayer for hepatotoxicity research in vitro.     

Cyclosporin A induces apoptosis in rat hepatocytes in culture

Cyclosporin A (CsA) at concentrations up to 1 μM induced apoptosis in a dose-dependent manner in cultured rat hepatocytes for 48 h in the presence of insulin and epidermal growth factor (EGF). The effect of CsA was evidenced by the DNA fragmentation pattern constituted by fragments of multiples of 180–200 base pairs, which is a characteristic of programmed cell death. The metabolic activity did not change significantly in the presence of 0.1 μM CsA and diminished to 49% of control in the presence of 1 μM CsA. Changes in the metabolic activity were correlated with a decrease in both [methyl-³H]thymidine uptake and DNA content, which reflects a decrease in the cell number. The treatment of cells with CsA (1 μM) decreased the metabolic activity/DNA content ratio by 24% with respect to dimethyl sulphoxide (DMSO) control, which also suggests, under these conditions, that the necrosis achieved is at most only 24%. In addition, the changes observed (apoptotic process, arrest of the cell cycle and apparition of a necrotic process) were correlated with an increase in the high-affinity guanosine triphosphatase (GTPase) enzymes. Received: 17 February 1998 / Accepted: 20 April 1998     

Metabolic activation and cytotoxicity of cyclophosphamide in primary cultures of postnatal rat hepatocytes

We have developed a model of primary cultures of postnatal rat hepatocytes to characterize the metabolic activation of xenobiotics to toxic intermediates and to study the mechanism(s) by which these chemicals produce cellular injury. This model was employed to investigate the cytochrome P-450 mediated biotransformation of cyclophosphamide (CP) to cytotoxic metabolites that nonspecifically alkylate DNA and cellular proteins. The parenchymal cells were isolated by an in situ collagenase perfusion technique and cultured for 24 hr prior to drug treatment. The cultures were then exposed to CP concentrations ranging from 1 × 10^?4 M to 1 × 10^?3 M for 24 hr. Initial studies indicated minimal toxicity to non-replicating parenchymal hepatocytes maintained in ornithine-supplemented, arginine-deficient medium. The addition of arginine permitted the overgrowth of fibroblasts in the same culture system. These fibroblasts then became the target of alkylating CP metabolites produced by the par-enchymal cells. By day 3 after CP administration, cell number and total protein per dish decreased by over 40 percent. The morphology of the cultures changed dramatically because of fibroblast destruction. The cytotoxicity to dividing fibroblasts was eliminated by administering 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF 525-A), an inhibitor of the cytochrome P-450 monooxygenase system, to the co-cultures treated with CP. The alkylating metabolites of CP produced by the parenchymal cells and released into the culture medium were quantitated by reacting aliquots of medium from CP-treated cells with 4-(p-nitrobenzyl)pyridine. These results provide both direct and indirect evidence of drug metabolism in cultured cells and suggest that this co-culture system can be utilized to evaluate the metabolic activation of xenobiotics.     

Study of embryotoxicity of Fusarium mycotoxin butenolide using a whole rat embryo culture model

Butenolide, a mycotoxin elaborated by several toxigenic Fusarium species, has been implicated as an etiological factor of Kashin-Beck disease and it is always detected in food from endemic Kashin-Beck disease areas. Although butenolide is considered as a potential health risk to humans and animals, its toxicity targets and mechanism of action have not been fully understood and the knowledge of its developmental toxicity is absent. The present study investigated butenolide embryotoxicity via an in vitro whole embryo culture system using rat embryos. Embryos exposed to butenolide at a concentration of 0.625 mg/L showed and differentiation similar to that of the control embryos (=no observed adverse effect concentration; NOAECwec). The embryonic growth and differentiation were affected, represented as reduced crown-rump length and head length, and decreased number of somites from 1.25 mg/L. Total morphological scores decreased significantly at the concentration of butenolide of 2.5 mg/L. All embryos were malformed at 3.75 mg/L and above (=ICMaxWEC), presenting growth retardation with flexion failure and irregular somite differentiation. The IC503T3 of butenolide as calculated from the balb/c 3T3 cytotoxicity test is 6.45 mg/L. Our study shows that butenolide exerts detrimental effects on embryo development in vitro by inducing growth retardation and differentiation inhibition, and the embryotoxicity effect of butenolide should be treated with caution.     

Tissue culture cytotoxicity assay for cyclophosphamide metabolites in rat body fluids

An in vitro cytotoxicity assay for cyclophosphamide metabolites in rat body fluids is described. Of the two tissue culture tumor cell lines employed, the Walker-256 rat carcinosarcoma was more sensitive to metabolite levels than the L-1210 mouse lymphocytic leukemia. The Walker-256 system detected cyclophosphamide metabolite levels two orders of magnitude lower than the commonly used 4-(p-nitrobenzyl)pyridine analytical procedure.     

Comparative evaluation of the teratogenicity of genistein and genistin using rat whole embryo culture and limbud micromass culture methods

Genistein (GEN) is one kind of phytoestrogen. Several studies have demonstrated the teratogenic potential of GEN in vitro by postimplantation rat whole embryo culture (WEC) assay, but GEN showed no teratogenic effects in vivo even at a dose up to 1000 mg/kg bw/day. The mechanism of such discrepancy is still unclear. Because more than 80% of total genistein (free plus glycoside form) in circulation is its glycoside metabolite, genistin (GIN), we thus hypothesize that genistin is non-teratogenic. To prove this hypothesis, rat whole embryo culture (WEC) and limbud micromass culture methods were applied to compare the teratogenic effects of GEN and GIN on developing embryos in vitro. In WEC assay, we found that the development of embryos was affected by GEN treatment dose-dependently, while GIN-treated embryos displayed slight developmental defects only at the highest dose (222 μM). In micromass culture assay, the IC50 of cell proliferation and differentiation for GEN were 15.6 and 37.2 μM, respectively, while neither was influenced by GIN treatment up to 111 μM. Collectively, our study indicated that GEN showed no teratogenic effects in vivo probably due to its transformation to the non-teratogenic metabolite, GIN.     

环磷酰胺与异环磷酰胺对离体大鼠肝细胞毒性机制

目的 探讨环磷酰胺（CP）与异环磷酰胺（Ifo)对混悬培养大鼠肝细胞的毒性效应及其可能机制。方法 以两步灌流法消化成年大鼠肝细胞,并进行混悬培养。CP与Ifo分别以20mmol/L染毒,观察染毒后30、60、120和180min肝细胞的存活率、胞内酶泄漏以及肝细胞巯基、MDA含量的变化,并对肝细胞表面形态和超微结构进行观察。结果 随着染毒时间的延长,两药均引起肝细胞存活率逐渐下降,胞内酶泄漏加重,增养液中乳酸脱氢酶（LDH）、天冬氨酸转移酶（AST）活性增高,同时肝细胞总巯基（TSH）、非蛋白巯基（NPSH）、蛋白巯基（PSH）也逐渐下降,其中PSH下降在TSH耗竭中起主要作用。两药引起的这些改变CP有比Ifo重的趋势,肝细胞MDA含量未发现有显著增遍,形态学检查发现CP与Ifo20mmol/L染毒180min均使肝细胞表面出现“大疱”,胞内线粒体肿胀,空泡化,粗面内质网扩张,部分脱颗粒,内腔模糊。结论 CP与Ifo对混悬培养大鼠肝细胞有损伤作用。巯基物质的降低在两药肝细胞毒性中起重要作用。