A New Sensitive Method for Quantitative Determination of Cisplatin in Biological Samples by Kinetic Spectrophotometry.

This study describes a kinetic spectrophotometric method for accurate, sensitive and rapid determination of cisplatin in biofluids. The developed method is based on the inhibitory effect of cisplatin on the oxidization of Janus Green by bromate in acidic media. The change in absorbance as the criteria of the oxidation reaction was followed spectrophotometrically. To obtain the highest rate of sensitivity, efficient reaction parameters were optimized. Under optimum experimental conditions, a calibration graph was obtained linearly over the range 10.0 - 5750.0 µg L-1 and the limit of detection (3sb/m) was 4.2 µg L-1 of cisplatin. The interfering effect of diverse species was investigated. The developed method was used for the quantification of cisplatin in bio fluids of patients treated with cisplatin, spiked bio fluids and pharmaceutical samples and yielded satisfactory results.

Nonallergenic urushiol derivatives inhibit the oxidation of unilamellar vesicles and of rat plasma induced by various radical generators.

Urushiols consist of an o-dihydroxybenzene (catechol) structure and an alkyl chain of 15 or 17 carbons in the 3-position of a benzene ring and are allergens found in the family Anacardiaceae. We synthesized various veratrole (1,2-dimethoxybenzene)-type and catechol-type urushiol derivatives that contained alkyl chains of various carbon atom lengths, including -H, -C1H3, -C5H11, -C10H21, -C15H31, and -C20H41, and investigated their contact hypersensitivities and antioxidative activities. 3-Decylcatechol and 3-pentadecylcatechol displayed contact hypersensitivity, but the other compounds did not induce an allergic reaction, when the ears of rats were sensitized by treatment with the compounds every day for 20 days. Catechol-type urushiol derivatives (CTUDs) exerted very high radical-scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl radical and inhibited lipid peroxidation in a methyl linoleate solution induced by 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). However, veratrole-type urushiol derivatives did not scavenge or inhibit lipid peroxidation. CTUDs also acted as effective inhibitors of lipid peroxidation of the egg yolk phosphatidylcholine large unilamellar vesicle (PC LUV) liposome system induced by various radical generators such as AMVN, 2,2'-azobis(2-amidino-propane) dihydrochloride, and copper ions, although their efficiencies differed slightly. In addition, CTUDs suppressed formation of cholesteryl ester hydroperoxides in rat blood plasma induced with copper ions. CTUDs containing more than five carbon atoms in the alkyl chain showed excellent lipophilicity in a n-octanol/water partition experiment. These compounds also exhibited high affinities to the liposome membrane using the ultrafiltration method of the PC LUV liposome system. Therefore, CTUDs seem to act as efficient antioxidative compounds against membranous lipid peroxidation owing to their localization in the phospholipid bilayer. These results suggest that nonallergenic CTUDs act as antioxidants to protect against oxidative damage of cellular and subcellular membranes.

A new VGLUT-specific potent inhibitor: pharmacophore of Brilliant Yellow.

The increased concentration of glutamate in synaptic vesicles, mediated by the vesicular glutamate transporter (VGLUT), is an initial vital step in glutamate synaptic transmission. Evidence indicates that aberrant overexpression of VGLUT is involved in certain pathophysiologies of the central nervous system. VGLUT is subject to inhibition by various types of agents. The most potent VGLUT-specific inhibitor currently known is Trypan Blue, which is highly charged, hence membrane-impermeable. We have sought a potent, VGLUT-specific agent amenable to easy modification to a membrane-permeable analog. We provide evidence that Brilliant Yellow exhibits potent, VGLUT-specific inhibition, with a Ki value of 12 nM. Based upon structure-activity relationship studies and molecular modeling, we have defined the potent inhibitory pharmacophore of Brilliant Yellow. This study provides new insight into development of a membrane-permeable agent to lead to specific blockade, with high potency, of accumulation of glutamate into synaptic vesicles in neurons.

Wall teichoic acid protects Staphylococcus aureus from inhibition by Congo red and other dyes.

OBJECTIVES: Polyanionic polymers, including lipoteichoic acid and wall teichoic acid, are important determinants of the charged character of the staphylococcal cell wall. This study was designed to investigate the extent to which teichoic acid contributes to protection from anionic azo dyes and to identify barriers to drug penetration for development of new antibiotics for multidrug-resistant Staphylococcus aureus infection. METHODS: We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of teichoic acid in vitro and in vivo. RESULTS: We observed that inhibition of wall teichoic acid expression resulted in an ∼1000-fold increase in susceptibility to azo dyes such as Congo red, reducing its MIC from >1024 to <4 mg/L. Sensitization occurred when the first step in the wall teichoic acid pathway, catalysed by TarO, was inhibited either by mutation or by chemical inhibition. In contrast, genetic blockade of lipoteichoic acid biosynthesis did not confer Congo red susceptibility. Based on this finding, combination therapy was tested using the highly synergistic combination of Congo red plus tunicamycin at sub-MIC concentrations (to inhibit wall teichoic acid biosynthesis). The combination rescued Caenorhabditis elegans from a lethal challenge of S. aureus. CONCLUSIONS: Our studies show that wall teichoic acid confers protection to S. aureus from anionic azo dyes and related compounds, and its inhibition raises the prospect of development of new combination therapies based on this inhibition.

Permeability changes in response to NONOate and NONOate prodrug derived nitric oxide in a blood-brain barrier model formed by primary porcine endothelial cells.

The influence of nitric oxide (NO) and NO-donors on the permeability of the blood-brain barrier (BBB) is still not well understood and the literature about this is quite controversial. Some studies suggest increasing, others decreasing or even no effects of NO-donors on the BBB permeability. In this work we report about the influence of three diazeniumdiolates, which release NO spontaneously and three different diazeniumdiolate prodrugs, which have to be cleaved chemically or enzymatically before releasing NO, on the permeability of an in vitro BBB-model formed by primary porcine endothelial cells. By measuring the flux of a small polar molecule (carboxyfluorescein: CF) we could show, that the NO-releasers PHEPIPERAZI/NO (sodium 1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-diolate), DBA/NO (sodium 1-(N,N-dibutylamino)diazen-1-ium-1,2-diolate) and DETA/NO (1-N,N-di-(2-aminoethyl)amino)diazen-1-ium-1,2-diolate) reduced the BBB-model permeability. In contrast, the NO-prodrugs Et-PHEPIPERAZI/NO (O(2)-Ethyl-1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-diolate) and TOSYL-PYRRO/NO (O(2)-(p-Methylbenzen-sulfonyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate) increased the permeability in all investigated concentrations, whereas the prodrug Et-BUPIPERAZI/NO (O(2)-Ethyl-1-(butylpiperazin-1-yl)diazen-1-ium-1,2-diolate) reduced it at the lowest investigated concentration of 100 microM, at the higher concentrations it increased the permeability. Blocking the effect of the BBB-model permeability reducing compounds could be done by methylene blue, whereas permeability increasing effects could not be blocked.

The interaction of flavonoids with membranes: potential determinant of flavonoid antioxidant effects.

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2'-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2'-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid-water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.

In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma.

Recently we identified four conjugated glucuronide metabolites of epicatechin, (-)-epicatechin-3'-O-glucuronide (E3'G), 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide (4'ME3'G), (-)-epicatechin-7-O-glucuronide (E7G) and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide (3'ME7G) from plasma and urine. E3'G and 4'ME3'G were isolated from human urine, while E7G and 3'ME7G were isolated from rats that had received oral administration of (-)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34,840-849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (-)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (-)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2'-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.

Influence of oligomer chain length on the antioxidant activity of procyanidins.

The antioxidant activity of catechin monomers and procyanidin (dimers to hexamers) fractions purified from cocoa was studied in two in vitro systems: liposomes and human LDL. Liposome oxidation (evaluated as formation of 2-thiobarbituric acid reactive substances) was initiated with 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN), iron/ascorbate, or UV-C; LDL oxidation (evaluated as formation of conjugated dienes) was initiated with Cu(2+) or AAPH. Catechin monomers and procyanidin fractions inhibited both liposome and LDL oxidation. Monomers, dimers, and trimers fractions were the most effective antioxidants when liposome oxidation was initiated in the aqueous phase. When oxidation was initiated in the lipid domains, higher molecular weight procyanidins were the most effective. All fractions significantly inhibited Cu-mediated LDL oxidation; no significant effect of procyanidin molecular weight was observed. The hexamer fraction was the least effective with respect to preventing AAPH initiated LDL oxidation. Results reported herein give further evidence on the influence of the oligomer chain length on the antioxidant protection by procyanidins.

Amphotericin B as an intracellular antioxidant: protection against 2,2'-azobis(2,4-dimethylvaleronitrile)-induced peroxidation of membrane phospholipids in rat aortic smooth muscle cells.

The antifungal activity of amphotericin B (AmB) and its side-effects (e.g. nephrotoxicity and hemolytic action) are suggested to be associated with its prooxidant effects in target cells. To test this hypothesis, we have undertaken studies to examine the role of AmB in oxidative stress in cultured rat aortic smooth muscle cells (SMC) incubated in the absence or in the presence of a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). No changes in the pattern of membrane phospholipids could be detected by two-dimensional high performance thin-layer chromatography (HPTLC) after oxidative stress induced by AMVN in which the cells remained viable, as judged by trypan blue exclusion. To improve the sensitivity of detection of oxidative stress in the cells, cis-parinaric acid (PnA) was incorporated biosynthetically into the membrane phospholipids [using PnA-human serum albumin (hSA) complex]. Incubation of the cells under aerobic conditions in the presence of up to 10 microM AmB showed no significant change in the pattern of PnA-labeled phospholipids, suggesting that AmB was not affecting the oxidative state of the cells. In contrast, treatment with AMVN (0.5 mM, incubation in the dark for 2 hr at 37 degrees--conditions in which the viability of the cells was maintained) caused a significant reduction of all fluorescently labeled phospholipid fractions separated by HPLC. When PnA-labeled cells were subjected to oxidative stress by incubation with 0.5 mM AMVN in the presence of AmB, the loss of fluorescent phospholipids was reduced in a concentration-dependent manner over a concentration range of 0.25 to 10 microM. Thus, AmB does not produce any prooxidant effect but rather acts as an intracellular antioxidant.

Tolerance to lipopolysaccharide-induced increase in vascular permeability in mouse skin.

We investigated whether tolerance develops to the lipopolysaccharide-induced increase in vascular permeability of mouse skin on pretreatment with Salmonella typhimurium lipopolysaccharide. Lipopolysaccharide-induced plasma extravasation was assessed by determining Pontamine sky blue dye accumulation in the skin where lipopolysaccharide was injected s.c. 2 h previously. When mice were pretreated with lipopolysaccharide (0.15 mg/kg i.p.), the dye leakage induced by s.c. challenge with lipopolysaccharide (400 micrograms/site) was significantly, inhibited for 2-24 h after pretreatment, indicating the development of lipopolysaccharide tolerance. At 4 h after lipopolysaccharide (0.15 mg/kg i.p.), the dose-response curve of dye leakage against the challenge dose of lipopolysaccharide shifted about 2-fold to the higher dose. The dye leakage induced by lipopolysaccharide was inhibited by pretreatment with lipopolysaccharide in a dose-dependent manner (0.05-0.15 mg/kg i.p.). Lipopolysaccharide tolerance was not seen in adrenalectomized mice. When mice were pretreated with lipopolysaccharide and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, at the same time, the hyporesponsiveness to lipopolysaccharide challenge disappeared. However, L-NAME was ineffective to inhibit the development of lipopolysaccharide tolerance when administered 24 h after lipopolysaccharide pretreatment or just before the lipopolysaccharide challenge. Tumor necrosis factor-alpha and interleukin-1 alpha but not interleukin-6 induced a similar hyporesponsiveness to lipopolysaccharide. These results suggest that tolerance develops to the lipopolysaccharide-induced increase in vascular permeability in mouse skin after a single lipopolysaccharide administration and that endogenous glucocorticoids and NO are necessary for induction of lipopolysaccharide tolerance. Hyporesponsiveness induced by lipopolysaccharide pretreatment may be mediated by production of some cytokines such as tumor necrosis factor-alpha or interleukin-1 alpha.

Hepatic damage induced by perfusion of radical generating azo compound and its inhibition by vitamin E.

The damaging effect of perfusion of hydrophilic radical generating azo compound on the liver of normal and vitamin E-deficient rats and its inhibition by antioxidants were studied in order to increase understanding of the action of free radicals on biological tissues. The hepatic damage was evaluated from the release of cytosolic enzymes such as glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, mitochondrial oxidation metabolism and morphological change. Two kinds of hydrophilic azo compounds were used, one which decomposes spontaneously at a uniform rate to generate free radicals and the other which does not. The former induced hepatic damage in a dose-dependent manner, while the latter did not exert any damage. Both endogenous vitamin E in the membranes and a water soluble vitamin E analogue added simultaneously with a radical initiator suppressed the hepatic damage. These results show that the hepatic damage induced by perfusion of radical generating azo compound is caused not by the azo compound itself but by free radicals.

Interaction of the pyridoindole stobadine with peroxyl, superoxide and chromanoxyl radicals.

The pyridoindole derivative stobadine [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido(4,3b)indole] has been described as a drug with antihypoxic and antiarrhythmic cardioprotective properties. Here its reactivity with peroxyl radicals in liposomes using a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azo-bis(2,4-dimethyl-valeronitrile) (AMVN), was examined. Stobadine exerted scavenging as evidenced by the inhibition of: (i) cis-parinaric acid fluorescence decay (half-maximal effect at 20 microM), or (ii) luminol-sensitized chemiluminescence (half-maximal effect at 33 microM). In rat liver microsomes, stobadine was equally efficient in inhibiting lipid peroxidation induced by lipid-soluble (AMVN) or water-soluble 2,2'-azo-bis(2-aminopropane)-HCl (AAPH), azo-initiators of peroxyl radicals with half-maximal effect at 17 microM. Stobadine partitions in a two-phase system (octanol-water) with the coefficient log P = 0.57 +/- 0.03, explaining its ability to quench peroxyl radicals in both lipid and aqueous phases. Stobadine is not an efficient scavenger of superoxide radicals. The second order rate constant for the reaction of stobadine with superoxide was estimated to be 7.5 x 10(2) M-1 sec-1 as measured by superoxide-induced lucigenin-amplified chemiluminescence. ESR measurements showed that stobadine in liposomes does not reduce the chromanoxyl radical of a vitamin E homologue with a 6-carbon side-chain, 2,5,7,8-tetramethyl-2-(4'-methylpentyl)chroman-6-ol(chromanol++ +-alpha-C6), in agreement with pulse-radiolysis results obtained using Trolox in homogeneous solution (Steenken et al., Chem Res Toxicol 5: 355-360, 1992). Stobadine increased the magnitude of the chromanoxyl and ascorbyl radical ESR signal generated by lipoxygenase+arachidonate. This was interpreted to be due to the interaction of stobadinyl radicals with the chromanol ring and ascorbate, respectively. It is suggested that high reactivity of stobadine radicals requires the presence of reducing antioxidants (vitamin E, vitamin C) to exhibit its antioxidant effects in physiological systems.

Inhibition of methylazoxymethanol acetate initiation of colon carcinogenesis in rats by treatment with the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

To clarify the biological role of poly(ADP-ribose) in cancer induction in vivo, the influence of the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3-AB), on initiation of carcinogenesis in the colon and liver by a single application of methylazoxymethanol (MAM) acetate was investigated. Since 3-AB is rapidly metabolized and excreted in vivo when injected as a single dose, rats were given a continuous i.v. infusion of the compound (1200 mg/kg/day) for 4 days and injected with a single dose (35 mg/kg) of MAM acetate 4 h after the start of the experiment. Rats were killed 70 weeks after the beginning of the experiment. The incidence of colon tumors was significantly lower (t less than 0.025) in the 3-AB-treated group than in the carcinogen-only controls. Although significant numbers of glutathione S-transferase placental form positive foci were also induced in the liver of MAM-acetate-treated animals, 3-AB administration had no effect on their number and size. The results thus clearly demonstrated that continuous infusion of 3-AB during the initiation phase inhibited the development of MAM-acetate-induced colon tumors, but was not effective for the formation of preneoplastic foci in the liver.

Retinol (vitamin A) inhibits the mutagenicity of o-aminoazotoluene activated by liver microsomes from several species in the Ames test.

Retinol (vitamin A) has earlier been shown to inhibit the mutagenicity of o-aminoazotoluene (OAAT) in the Salmonella/microsome assay when OAAT is activated with S9 from Sprague-Dawley rats. The results presented in this paper confirm this and also show that S9 from mice, hamsters and gerbils activates OAAT to mutagenic metabolites detected by Salmonella typhimurium TA100. However, S9 from rabbits is inactive. The S9 fraction from rabbits also shows a low aryl hydrocarbon hydroxylase (AHH) activity. The AHH activity or protein content of the microsomal fraction cannot be used to predict the activating capacity of S9 from the other species. Retinol, added in vitro, inhibits the mutagenic effect of OAAT activated by mouse, gerbil or hamster S9. The strongest inhibition is observed with hamster S9 while the inhibition of mouse and gerbil S9 is lower but still higher than in the rat.

Importance of the duration of inhibition on intestinal carcinogenesis by difluoromethylornithine in rats.

The effect of the duration and sequence of inhibition of intestinal tumor formation in rats was studied to determine whether part time inhibition has any value. Four groups of male Sprague-Dawley rats were given 8 weekly s.c. injections of azoxymethane (AOM) 8 mg/rat. Three groups were given the inhibitor, difluoromethylornithine (DFMO) in the drinking water; one for the entire 26 weeks of the study, one for the first 13 weeks only, and one for the last 13 weeks. A control group was not given the inhibitor. While the continuous treatment group developed the least number of tumors per rat (1.5 vs. 5 for controls), still both groups given the inhibitor for just 13 weeks also developed fewer tumors than controls 5 vs. 3.2 (early treatment) and 5 vs. 2.8 (late treatment). These results show that part time inhibition, including its late application, does reduce intestinal tumor formation in rats.

Effect of dietary butylated hydroxyanisole on methylazoxymethanol acetate-induced toxicity in mice.

Administration of butylated hydroxyanisole (BHA), a widely used food additive, has been found to inhibit the carcinogenic and toxic effects of various chemicals in animal models. To study the relationship of dietary BHA to the acute toxicity of methylazoxymethanol (MAM) acetate, a colon-specific carcinogenic compound, groups of female CF1 mice were fed NIN-07 diet containing 0, 300, 1000, 3000 or 6000 ppm BHA or a semipurified diet containing 0 or 6000 ppm BHA for 4 wk, and were injected ip with MAM acetate (20 mg/kg body weight) at the end of the first 2 wk and again 4 days later. At levels of 300-6000 ppm, BHA was found to protect against death caused by MAM acetate. The mortality rates in MAM-treated mice were 80 and 92% in those fed the diets with no BHA and 0 and 1% in those fed 6000 ppm BHA, and were inversely related to the amount of BHA in the diet. The protection was associated with increased levels of hepatic cytochrome P-450 and b5 and with a reduction in necrotic changes in the liver.

Resonance Raman spectroscopy of arsanilazocarboxypeptidase A: mode of inhibitor binding and active-site topography.

The interaction of inhibitors with the active site of arsanilazocarboxypeptidase A has been investigated by means of resonance Raman spectroscopy. The resonance Raman bands of the active-site azotyrosine-248 residue have been shown previously to be sensitive to its state of ionization and its interactions with nearby groups. In particular, the azophenol form of azotyrosine-248 can adopt two different coexisting conformations that differ with respect to the presence or absence of an intramolecular hydrogen bond between the phenolic proton and a nitrogen atom of the azo group. Each of these conformations exhibits characteristic vNN and v phi N azo stretching frequencies. The relative concentrations of these two forms, revealed by resonance Raman spectroscopy, are a sensitive probe of the hydrogen bond accepting ability of the local environment. The present study shows that the binding of L-benzylsuccinate, phenylacetate, L-phenyllactate, and beta-phenylpropionate markedly perturbs the distribution of the intra- and intermolecularly hydrogen-bonded forms of azotyrosine-248 in water. In contrast, glycyl-L-tyrosine and L-phenylalanine leave this distribution unperturbed. These results, taken jointly with other data on inhibitor binding, serve to identify common binding sites for groups of inhibitors and result in plausible suggestions concerning the interactions between azotyrosine-248 and these inhibitors that lead to binding.

[Antidiabetogenic activity of maninyl]. / O protivodiabetogennom deistvii maninila.

Experiments were conducted on rabbits. A study was made of the effect of administration of maninyl (glybenclamide) into the stomach in a dose of 10 mg/kg of body weight for 7 days on the blood glucose level, insulin and zinc content in the pancreatic islands, and on the "dithizone" diabetes development. Maninyl administration was accompanied by a significant glycemia reduction. The amount of deposited insulin and zinc determined histochemically was sharply reduced up to complete disappearance from the majority of beta-cells. "Dithizone" diabetes was not reproducible in animals given maninyl preliminarily: the required condition for induction of this affection was formation of zinc dithizonate in beta-cells.

Enhancing and inhibitory effects of some stilbene and steroid compounds on induction of hepatoma in rats fed 3'-methyl-4-(dimethylamino)azobenzene.

Examinations were made on substances that enhance or inhibit the induction of hepatoma in rats previously fed 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) for a brief period. The substances tested were stilbene, 4-nitrostilbene, 4,4'-dihydroxystilbene, diethylstilbestrol, 17beta-estradiol, and methyltestosterone. Male Donryu rats were fed 0.5 g of 3'-Me-DAB by being maintained on a diet containing 0.06% 3'-Me-DAB, and then they were fed 0.25 or 0.5 g of a test substance with the basal diet. Comparison of the development and yield of hepatomas indicated that 4-nitrostilbene and methyltestosterone had an activity of enhancing 3'-Me-DAB carcinogenesis, whereas diethylstilbestrol and 17beta-estradiol had an activity to retard it. Other substances showed no such activities. The enhancement by 4-nitrostilbene and inhibition by diethylstilbestrol of 3'-Me-DAB carcinogenesis was correlated with their effect on liver nucleic acid metabolism. Feeding of 4-nitrostilbene caused a selective inhibition of Mn2+-(NH4)2SO4-activated RNA polymerase activity of liver nuclei and reduced liver RNA content. The deleterious alteration of liver RNA metabolism was followed by the enhancement in the incorporation of ip-injected 3H-thymidine into DNA of liver nuclei. On the other hand, feeding of diethylstilbestrol increased tissue RNA content without effect on RNA polymerase activity of liver nuclei, and had an activity of increasing the incorporation of 3H-thymidine into DNA. The possible implication of these results with regard to the enhancement and inhibition of hepatocarcinogenesis is discussed.