In vitro transformation of hamster embryo cells by quercetin

In vitro carcinogenic activities of bracken extract and two flavone derivatives, kaempferol and quercetin were examined in a transformation assay with cryopreserved hamster embryo cells. Bracken extract and kaempferol did not induce transformation. But quercetin induced morphological transformation of the cells at the concentration of 5 and 10 .     

Inhibition of chemical transformation of hamster embryo cells by retinoids

13-cis-Retinoic acid, trans-retinoic acid, 13-cis-retinal, trans-Tetinal and trans-retinol inhibited morphological transformation of hamster embryo cells induced by N-butyl-N-acetoxymethylnitrosamine (BAMN). Addition of 13-cis-retinoic acid after 24-h treatment with N-butyl-N-acetoxymethylnitrosamine also inhibited induction of transformation, suggesting that retinoids inhibited the progressive stage of transformation. The five retinoids tested all altered the orientation of the cells and enhanced density dependent inhibition. Hamster embryo cells treated with 13-cis-retinoic acid could be stained with periodic acid-Schiff base staining, while untreated cells could not.     

In vitro transformation of hamster embryo cells with a glutamic acid pyrolysis product

The in vitro activity of the glutamic acid pyrolysis product 2-amino-6-methyl-dipyrido[l,2-a:3',2'-d]imidazole (Glu-P-1) was examined in a transformation assay with cryopreserved hamster embryo cells. Glu-P-1 was cytotoxic at a concentration of 50 μg/ml, but induced morphological transformation of the cells at concentrations of 10 μg/ml and 20 μg/ml. Glu-P-1 may be carcinogenic for experimental animals.     

Statistical evaluation of Pienta's in vitro carcinogenesis assay

The in vitro carcinogenesis assay developed by Pienta was statistically evaluated. Transformation assay of 3-methylcholanthrene (3MC) and solvent control were repeated 8 times. There was no transformation in controls and 3MC induced 7.38 transformed colonies as an average. Fitting the binomial distribution the possibility to take a false negative was calculated. It is concluded that the positive result is highly reliable but there is considerably high risk to take a false negative unless experiments are repeated.     

Morphological transformation of hamster embryo cells by cancer chemotherapeutic agents

Studies were made on induction of morphological transformation of hamster embryo cells by various types of cancer chemotherapeutic agents, including alkylating agents, antimetabolites, antibiotics and natural products. Cyclophosphamide, actinomycin D, mitomycin C and adriamycin hydrochloride, which are widely used as cancer chemotherapeutic agents and are also known to be carcinogenic in animals, produced significant morphological transformation. Two other compounds that are known to be carcinogenic in vivo, thio-TEPA and daunomycin hydrochloride, did not induce transformation. The anti-metabolites 6-mercaptopurine, 5-fluorouracil, cytosine arabinoside and methotrexate also did not produce morphological transformation of hamster embryo cells.     

Transformation of hamster embryo cells by neutral sterols and bile acids

Toxicity and cell transformation of Syrian hamster embryo cells in culture by certain neutral sterols and bile acids show interesting trends related to their structures: cholesterol-α-epoxide and cholestan-3β,5α,6β-triol were more toxic and induced transformation to these cells, whereas their metabolic precursor, cholesterol, was inactive. The secondary bile acids, lithocholic and deoxycholic acids, were more toxic than their primary bile acid precursors, cholic and chenodeoxycholic acids and transformed the cells. These data suggest that mammalian cell transformation is a useful short-term assay to measure the potential toxicity and carcinogenicity of steroid derivatives.     

In vitro metabolism of penicillium roqueforti toxin (PRT) and a structurally related compound, eremofortin A, by rat liver.

The metabolism of two closely related compounds (PRT and Eremofortin A) isolated from a culture of Penicillium roqueforti was studied in vitro using the rat liver system. Enzymatic activity was enhanced by treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC). Metabolites were isolated by silica gel column chromatography and their structures elucidated by spectral analysis and confirmed by chemical synthesis.Three types of enzymatic reactions were observed: hydroxylation, reduction and deacetylation; the metabolic pathways of the two compounds give the same final product. The authors suggest that the pathway described here is a route of detoxication.     

Effects of tobacco smoke compounds on the ciliary activity of the embryo chicken trachea in vitro

The ciliotoxicity of 316 individual compounds representative of the gaseous and semivolatile phases of tobacco smoke has been investigated using chicken tracheal organ cultures. When examined at 5 mM concentration and measuring the time to complete ciliostasis, 36% of the compounds were found to cause ciliostasis within 15 min, while about 50% had no visible effect on the ciliary activity during a 60-min exposure. The majority of the ciliotoxic compounds were either alkylated phenylethers, benzonitriles, benzaldehydes, phenols, benzenes, naphthalenes and indoles, or α,β-unsaturated ketones and aldehydes or C₆C₁₀ aliphatic alcohols, aldehydes, acids and nitriles. Most of the compounds classified as benzoic acids, esters, polyaromatic hydrocarbons, amines and N-heterocycles, except indoles, were found to be inactive.     

In vivo and in vitro effects of 3-methylcholanthrene on the microsome-mediated in vitro mutagenicity of 2-acetylaminofluorene

Pretreatment of rat, hamster or mouse by 3-methylcholanthrene (3-MC) largely induces the liver microsomal N-hydroxylase activity. The same pretreatment given simultaneously with 2-acetylaminofluorene (2-AAF) inhibits the hepatocarcinogenicity in the rat but not in the hamster.The present report compares the in vivo and in vitro effects of 3-MC on liver microsomal N-hydroxylation and liver microsome-mediated mutagenicity of 2-AAF in hamster, rat and mouse. The induction of hamster or mouse liver microsomal N-hydroxylase activity correlates well with the increase in the microsome-mediated mutagenicity of 2-AAF. With rat, however, even though the N-hydroxylase activity is largely enhanced, microsome-mediated mutagenicity is significantly reduced after pretreatment with 3-MC. Such a reduction parallels a decrease in enzyme affinity.Added in vitro to the incubation medium, 3-MC (μM concentration) inhibits both the N-hydroxylase activity and the microsome-mediated mutagenicity of 2-AAF. Those data are discussed in relationship with the biological interactions between 3-MC and 2-AAF.     

In vitro transformation of hamster embryo cells by 3-(N-salicyloyl) amino-1,2,4-triazole

The in vitro carcinogenic activities of 3-(N-salicyloyl)amino-1,2,4-triazole (SAT) and its two components, 3-amino-1,2,4-triazole and phenyl salicylate were examined in a transformation assay with cryopreserved hamster embryo cells. SAT induced morphological transformation at certain doses between 10 $\text{μg}\text{ml}$ and 100 $\text{μg}\text{ml}$ in each of 5 repeated experiments, but gave a negative result in the Salmonella mutation assay. 3-Amino-1,2,4-triazole also induced transformation in 3 repeated experiments. Phenyl salicylate did not induce transformation at any of the doses tested in 3 consecutive experiments.     

Covalent binding of benzo[a]pyrene to rat liver cytosolic proteins and its effect on the binding to microsomal proteins

Benzo[a]pyrene will bind covalently to rat liver cytosolic proteins when incubated with microsomes and NADPH. The binding is most extensive when microsomes from 3-methylcholanthrene-treated rather than phenobarbital-treated or control rats are used. The binding to cytosolic proteins increases when incubations are performed with increasing concentrations of cytosol. At the same time the covalent binding of benzo[a]pyrene to microsomal proteins decreases. Two cytosolic polypeptides are the main targets for benzo[a]pyrene. These have the same mobility in polyacrylamide gels as the subunits of purified glutathione S-transferase B. These subunits also react covalently with benzo[a]pyrene when the transferase is incubated with microsomes and NADPH.     

Interactions of styrene and styrene oxide with partially purified cytochrome P-450 and P-448 from rat liver microsomes

Styrene and styrene 7,8-oxide were able to bind both to partially purified cytochrome P-450 isolated from phenobarbital (PB)-treated rat liver and to cytochrome P-448 from liver microsomes of 3-methylcholanthrene (3-MC)-treated rats.In the presence of either purified preparation or “fresh” microsomes from PB- or 3-MC-treated animals, styrene produced a characteristic Type I difference spectrum as did styrene 7,8-oxide with “fresh” microsomes from PB rats. In other experiments, the addition of styrene oxide produced spectra which resembled Type I spectra but were somewhat shifted to longer wavelengths.A comparison of the binding parameters for the interaction of styrene or styrene 7,8-oxide with partially purified preparations and “fresh” microsomes indicated that the binding is catalyzed by more than one type of P-450 hemoprotein and that the binding affinity is slightly reduced by the purification procedure. The addition of phosphatidylcholine was unable to restore the binding parameters.     

In vitro mutagenicity of rubber chemicals and their nitrosation products

14 chemicals employed in rubber manufacture were assayed in the Salmonella reversion test with the strains TA98 and TA100. Mixed diaryl-p-phenylenediamines were weakly mutagenic in TA98 after metabolic activation; poly-p-dinitrosobenzene was active in TA98 without as well as with S9. After in vitro reaction with nitrite at low pH, mixed diaryl-p-phenylenediamines became directly mutagenic in both strains, whereas poly-p-dinitrosobenzene retained its activity unchanged. Furthermore, 4 of the remaining chemicals acquired mutagenic characteristics: in the presence of S9, N,N'-dimethylpentyl-p-phenylenediamine reverted TA98 and hexamethylenetetramine reverted both TA98 and TA100; N-isopropyl-N'-phenyl-p-phenylenediamine was mutagenic in TA98 with and without S9; N-nitrosodiphenylamine was active in both strains without S9 and weakly mutagenic in TA98 after metabolic conversion.     

Kinetics of in vitro O-deethylation of phenacetin and 7-ethoxycoumarin by rat intestinal mucosal cells and microsomes. The effect of induction with 3-methylcholanthrene and inhibition with alpha-naphthoflavone

A novel, sensitive (0.5 ng) assay for acetaminophen, using HPLC with selective electro-chemical detection, enabled us to study rat small intestinal O-deethylation of phenacetin and compare it with corresponding 7-ethoxycoumarin-O-deethylation. Two in vitro systems, i.e. isolated intestinal mucosal cells and microsomal fractions thereof, were used to study kinetics for the O-deethylation of both substrates. Kapp m- and Vmax-values are similar for 7-ethoxycoumarin- and phenacetin-O-deethylation. Apparent Km-values varied between 50 and 70 microM in control rats and decreased after 3-methylcholanthrene pretreatment to 20-45 microM. Vmax-values were increased by 3-methylcholanthrene pretreatment. O-Deethylation was inhibited equally in cells and microsomes by alpha-naphthoflavone, but is inhibited more markedly in intestinal preparations after pretreatment with 3-methylcholanthrene. It is suggested that 7-ethoxycoumarin and phenacetin are O-deethylated by different forms of cytochrome P-450 with almost identical Kapp m and that these enzymes have a different distribution along the villus.     

Influence of red blood cells,serum albumin,and serum lipoproteins on the clearance of benzo[a]pyrene by isolated livers of 3-methylcholanthrene-treated rats

Red blood cells, serum albumin, and serum lipoproteins transport benzo[a]pyrene and other xenobiotic compounds in the circulation. The distribution of benzo[a]pyrene and its metabolites among these blood components was examined, and the effect of their presence in the perfusion medium on the ability of isolated livers from 3-methylcholanthrene-pretreated rats to clear circulating benzo[a]pyrene was determined. A large fraction (45%) of the benzo[a]pyrene in rat blood was associated with the serum lipoproteins. However, only 8% of the benzo[a]pyrene metabolites was associated with this component. Forty to forty-five percent of each was associated with red blood cells. Benzo[a]pyrene clearance by isolated rat livers was 1.8 ± 0.2 ml/min when the medium contained only red blood cells and buffer. Addition of serum lipoproteins or serum albumin increased benzo[a]pyrene clearance to 5.1 ± 0.5 or 8.5 ± 0.9 ml/min respectively. Appearance of benzo[a]pyrene metabolites in perfusion medium and bile was similarly altered by the changes in medium composition. These results indicate that the clearance of benzo[a]pyrene by rat liver depends on the composition of the medium perfusing the organ and suggest that alterations in blood components in vivo may influence the metabolic disposition of this carcinogen.     

Studies in the metabolism of carcinogenic polycyclic heteroaromatic compounds. I. The hepatic microsomal metabolism of 7-methylbenz[c]acridine

An enzyme assay for the metabolism of the carcinogenic aza-aromatic polycyclic compound 7-methylbenz[c]acridine has been developed using a modification of a radiochemical assay described for the polycyclic aromatic hydrocarbon benzo[a]pyrene by DePierre et al. [J. W. DePierre, M. S. Moron, K. A. M. Johannesen and L. Ernster, Analyt. Biochem. 63, 470 (1975)]and Van Cantfort et al. [J. Van Cantfort, J. DeGraeve and J. E. Gielen, Biochem. biophys. Res. Commun. 79, 505 (1977)]. When the activities of control microsomes and microsomes of phenobarbital-, 3-methylcholanthrene-and 7-methylbenz[c]acridine-pretreated animals were compared, strong similarities were displayed toward oxidation of benzo[a]pyrene and 7-methylbenz[c]acridine. These similarities were seen in turnover numbers, Michaelis constants, and inducibility of both enzyme systems. 7-Methylbenz[c]acridine afforded a type I difference spectrum with 3-methylcholanthrene-pretreated microsomes. It is suggested that 7-methylbenz[c]acridine is oxidized by the same or a similar set of enzymes which is responsible for benzo[a]pyrene metabolism.     

Factors influencing the microsome- and mitochondria-catalyzed in vitro binding of diethylnitrosamine and N-nitrosopiperidine to deoxyribonucleic acid.

[¹⁴C]Diethylnitrosamine ([¹⁴C]DEN) and [¹⁴C]N-nitrosopiperidine ([¹⁴]NPiP) bind covalently to calf thymus DNA in an in vitro incubation system containing rat liver microsomes. The reaction is NADPH-dependent. Pretreatment of the animals with phenobarbital (PB) enchances the binding of both DEN and NPiP to DNA, whereas the binding of DEN to DNA decreases after 3-methylcholanthrene pretreatment. The PB effect, as observed from the binding of DEN to DNA. is more pronounced in young rats than in the older animals. Addition of cytosol to the incubation system enhances the binding of DEN 3- to 4-fold and the binding of NPiP 2- to 3-fold. Addition of mitochondria to the incubation system increases the binding of [¹⁴C]DEN only slighty. but increases the binding of NPiP more than 5-fold. Addition of mitochondria has no effect on the binding of [¹⁴C]dimethylnitrosamine ([¹⁴C]DMN). Mitochondria alone markedly catalyze the binding of NPiP to DNA. Addition of benzylamine. which is a substrate of mitochondrial monoamine oxidase as well as an inhibitor of DMN-demethylase, inhibits the binding of NPiP catalyzed by microsomes and microsomes plus mitochondria.     

Correlation of mutagenicity and 4-(p-nitrobenzyl)-pyridine alkylation by epoxides

The reactivity of simple epoxides with 4-(p-nitrobenzyl)-pyridine was compared with their mutagenicity in Salmonella typhimurium TA 100 and Escherichia coli WP 2 uvrA. The order of reactivity correlated well with mutagenicity, trichloropropylene oxide being most potent followed by epichlorohydrin, styrene oxide, glycidol and propylene oxide. The results suggest that 4-(p-nitrobenzyl)-pyridine alkylation is a simple and reliable primary assay in the evaluation of mutagenic properties.     

环磷酰胺诱发叙利亚地鼠胚胎细胞的恶性转化

给予３７．５，７５和１５０ｍｇ·Ｌ－１环磷酰胺（ＣＰ）均可稳定地引起叙利亚地鼠胚胎细胞的表型转化．转化细胞在传至９９代时，仍然具有旺盛的分裂增殖能力，对细胞生长因子的依赖程度下降，转化细胞可因植物凝集素的存在而发生凝集，具有在软琼脂培养基上形成集落的能力，表现出抛锚独立性生长特性；接种同种动物及无胸腺裸鼠后能形成肿瘤．瘤切片病理观察显示所形成肿瘤均为分化程度较高的纤维肉瘤，从而证明ＣＰ能够诱导叙利亚地鼠胚胎细胞发生恶性转化．