[Mutagenic Activity of Four Aminoazo Compounds with Different Carcinogenicity for Rat Liver in the Ames Test].

In this paper in the bacterial Ames test we compared the mutagenicity of four aminoazo compounds, previously studied by other researchers and used for activation of rat liver enzymes, with the carcinogenicity in the rat liver. It was found that in the Ames test they have mutagenic activity, however, this activity does not correlate quantitatively with rat sensitivity to their hepatocarcinogenic action. Thus, the most active carcinogen 3'-methyl-4-dimethylaminoazobenzene causes mutations almost 2.5 times less than weakly carcinogenic ortho-aminoazotoluene, and exactly the same number of mutations as non-carcinogenic N,N-diethyl-4-aminoazobenzene.

[Mutagenic activation and carcinogenicity of aminoazo dyes of ortho-aminoazotoluene and 3'-methyl-4-dimethylaminoazobenzene in experiments on suckling mice].

It is found that after administration of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB,) which was hepatocarcinogenic to rats, in suckling mice, the number of neoplastic lesions in the liver of mice was 3 times higher than after analogous administration of equimolar dose of ortho-aminoazotoluene (OAT)). However, in the Ames test (TA-98 strain of Salmonella typhimurium) with activation by hepatic enzymes (S-9 fraction) of both intact and Aroclor-1254-induced mice and rats OAT contributed by an order of magnitude to revertant colonies compared to 3'-Me-DAB. In vivo inhibition of sulfotransferase activity, the enzyme which catalyzes the final stage of the mutagenic activation of aminoazo dyes, had no effect on carcinogenicity of 3'-Me-DAB but more than 4 times elevated that of OAT. It was concluded that the mechanism of carcinogenic action of aminoazo dyes studied is not genotoxic and that the carcinogenic potential of OAT is lost in the process of mutagenic activation.

Chronic alcohol consumption prevents 8-hydroxyguanine accumulation in 3'-methyl-4-dimethylaminoazobenzene-treated mouse liver.

Alcohol consumption is known to have opposing effects on carcinogenesis: promotion and prevention. In this study, we examined the effects of 12% ethanol on oxidative DNA damage accumulation and its repair in mouse livers treated with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), a well-known hepatic carcinogen. We previously reported that 3'-MeDAB increased 8-hydroxyguanine (8-OH-Gua) accumulation and its repair activity, accompanied by the fragmentation of 8-oxoguanine DNA glycosylase 1 (OGG1), the main repair enzyme of 8-OH-Gua. The present results showed that 12% ethanol intake attenuated the 8-OH-Gua accumulation, but not the fragmentation of OGG1 induced by 3'-MeDAB. Additionally, no significant changes in oxidative status, as monitored by lipid peroxidation (LPO), were observed among the 3'-MeDAB-treated mouse livers with/without alcohol administration. These findings suggested that 12% ethanol consumption may reduce the risk of 3'-MeDAB-induced carcinogenesis by decreasing 8-OH-Gua accumulation.

Expression of retinoid X receptor alpha is decreased in 3'-methyl-4-dimethylaminoazobenzene-induced hepatocellular carcinoma in rats.

The identification of the specific molecular targets, which underlie liver carcinogenesis is essential for the establishment of an effective strategy for the prevention and/or treatment of hepatocellular carcinomas (HCCs). We previously found that a malfunction of RXRalpha due to its aberrant phosphorylation was associated with the development of HCCs. However, it has remained unclear whether the abnormalities in the expression of RXRalpha or the other retinoid receptors play a role in the early stage of liver carcinogenesis. The present study was designed to determine whether alterations in the expression of RXRalpha and the other retinoid receptors RARalpha and RARbeta are involved in hepatocarcinogenesis using a 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-induced rat liver carcinogenesis model. We found that immunohistochemical expression of RXRalpha was decreased in liver cell tumors (HCCs and adenoma) and glutathione S-transferase placental form (GST-P)-positive foci, which is a precancerous lesion of HCC, when compared with the non-cancerous tissues. Western blot and RT-PCR analyses revealed a progressive decrease in the expression levels of RXRalpha, RARalpha, and RARbeta proteins and their mRNAs in 3'-MeDAB-induced HCCs and their surrounding tissues, when compared with the normal liver tissues from the control group. Moreover, the expression level of beta-catenin, the heterodimeric partner for both RXRalpha and RARalpha, was immunohistochemically observed in the cytoplasm and, in some cases, in the nucleus of HCC cells. The nuclear expression of cyclin D1, the downstream target molecule of beta-catenin, was also increased in HCC cells when compared with their adjacent normal appearing tissues. Our findings suggest that loss of retinoid receptors, especially RXRalpha, plays a critical role in the chemically-induced rat liver carcinogenesis and this might be associated with the activation of beta-catenin-related signaling pathway.

Advanced health assessment in nurse practitioner programs: follow-up study.

The increase in advanced practice graduate programs and the inclusion of content and skills related to advanced health assessment as a core competency for practice served as the impetus for a 5-year follow-up study to track the changes, methodologies, and integration of technology into practitioner programs. The questionnaire was mailed to the faculty/schools listed as current members in the National Health Service Corps Nurse Practitioner Faculty Advocate Network. The number of responding schools was 135 (44%). The family nurse practitioner program continues to be the most offered advanced practice nursing program. Nearly all institutions offer a post-master's program and an advanced health assessment course to their clinical graduate students. Health assessment is usually taught concurrently or as a prerequisite for clinical experiences; there continues to be a strong emphasis on the physical examination component. Ethnic and cultural assessment and gerontological assessment content increased since the original study. Both class and laboratory class sizes decreased. Qualitative data that centered on differences in graduate versus undergraduate health assessment revealed a shift in focus in several areas: differential diagnoses, abnormals, and the inclusion of advanced skills. There was an emergence of more creative strategies: the use of standardized patients, online coursework, videotaping, "live" patients, and simulations.

Hepatocarcinogenic activities of hydroxymethyl derivatives of 4-(N,N-dimethylamino)azobenzene in ACI/N rats.

The hepatocarcinogenic potencies of three newly identified hydroxymethyl derivatives of 4-(N,N-dimethylamino)azobenzene [(DAB) CAS: 60-11-7], i.e., 2'-CH2OH-DAB, 3'-CH2OH-DAB, and 4'-CH2OH-DAB, were strictly evaluated in a long-term test (400 days) and compared to the potency of 3'-CH3-DAB. ACI/N rats, known to be less sensitive to azo dye carcinogenesis, were given one of these compounds in their diets for 120 days. The incidence of hepatocellular carcinoma in group 2 (20/20), which was given 3'-CH2OH-DAB, was much higher than that in any of the other groups: group 1 (2'-CH2OH-DAB; 4/19), group 3 (4'-CH2OH-DAB; 1/25), or group 4 (3'-CH3-DAB; 3/24). These data suggest that 3'-CH2OH-DAB is the most potent hepatocarcinogen in the series of azo dyes. Possible reasons for the potency of the chemical are discussed.

Persistently decreased hepatic levels of 5'-deoxy-5'-methylthioadenosine during regeneration of and chemical carcinogenesis in rat liver.

The hepatic levels of 5'-deoxy-5'-methylthioadenosine (MTA) were measured in the livers of adult male Sprague-Dawley rats a) killed at various times during the liver regeneration process, b) killed at times after partial hepatectomy when the liver mass had already been completely restored (hereafter called post-regeneration livers), or c) continuously fed 3'-methyl-4-dimethyl-aminoazobenzene (CAS: 55-80-1) up to the full development of hepatoma and killed at regular intervals during hepatocarcinogenesis. Hepatic MTA levels were always significantly decreased, although to different degrees in both in vivo models of hepatic growth and at all times during the investigation. Astonishingly, the MTA levels were also significantly decreased in the post-regeneration livers, in which there was also a significant increase in the activity of adenosylmethionine decarboxylase (S-adenosyl-L-methionine decarboxylase; EC 4.1.1.50) with normal levels of activity of ornithine decarboxylase (EC 4.1.1.17). These results demonstrate that a) the MTA content is always decreased in rat liver whenever this organ is involved in a proliferative process (whether controlled or uncontrolled); b) the decrease in hepatic MTA content is a biochemical feature necessary for, but by no means by itself sufficient for, hepatocyte proliferation to occur, since this decrease remains long after complete restoration of the liver mass; and c) the return of the hepatocytes to the normal biochemical program after restoration of the liver mass is not complete, even though these cells become quiescent, because there are still some biochemical abnormalities in the post-regeneration livers.

Inhibitory effect of fumaric acid on hepatocarcinogenesis by thioacetamide in rats.

An inhibitory effect of fumaric acid (FA) on hepatocarcinogenesis was examined in rats fed thioacetamide (TAA). A group of male DONRYU rats were fed TAA at a level of 0.035% in the diet for 40 weeks and then fed a basal diet for 40 weeks. Hepatic carcinomas developed in 9 of 41 animals of this group fed TAA alone. The effect of FA on the carcinogenesis was examined in 2 groups fed both TAA and FA; one group of rats were fed FA at 1% in a basal diet after ingestion of TAA, and another group of rats were fed TAA plus a supplement of 1% FA in the diet. The inhibitory effect of FA on TAA carcinogenesis was so marked that no hepatic carcinomas were found in both groups fed FA in combination with TAA.

gamma-Glutamyltransferase and the inhibition of azo dye-produced neoplasia by concomitant administration of disulfiram.

The concentration and total amount of DNA in the livers of SD rats fed 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) gradually increased and reached a maximum in developing tumors. In SD rats fed 3'-Me-DAB plus disulfiram (DSF), the concentration of DNA was higher than in controls, but it soon became stabilzed and the total amount of DNA in the liver did not differ substantially from that in rats fed DSF alone. In rats given 3'-Me-DAB, neoplastic nodules and liver carcinomas appeared after 3 months, but in those fed both compounds these formations were absent even after 6 months. The activity of gamma-glutamyltransferase (GT-ase), a marker of chemically induced carcinogenesis in rat liver, gradually increased to extremely high levels in tumors even after 75 days when the diet of the animals was changed to a normal one. In rats fed 3'-Me-DAB plus DSF, GT-ase activity increased for the greater part of 80 days, gradually leveled off around the 100th day, and returned to almost normal levels when the rats were given a normal diet after 100 days. We concluded that DSF 1) did not interfere with 3'-Me-DAB-induced proliferation of preneoplastic cells and the increase in GT-ase associated with this reversible adaptation to the influx of 3'-Me-DAB; and 2) inhibited malignant transformation and, consequently, prevented the formation and proliferation of neoplastic cells and the increase in constitutive GT-ase related to neoplasia.

Inhibitory effect of fumaric acid on 3-methyl-4'-(dimethylamino)-azobenzene-induced hepatocarcinogenesis in rats.

Fumaric acid (FA) was examined for its effect on hepatocarcinogenesis in rats fed 3-methyl-4'-(dimethylamino)azobenzene (3-Me-DAB). Male DONRYU rats were given approximately 0.5 g 3-Me-DAB by being fed a diet containing 0.06% 3-Me-DAB for 50 days; they were then given a diet containing 1% FA and drinking water containing 0.025% FA for 51 weeks. The administration of FA effectively suppressed the development of hepatocellular carcinoma, hyperplastic nodules, and hyperplastic areas in the livers of rats fed 3-Me-DAB.

Antitumor activity of vitamin A and its derivatives.

Studies were conducted for investigation of the inhibitory effect on the development of experimental tumors of the skin and liver with vitamin A-like compounds, with a particular focus on a new synthetic derivative of the polyprenoic acid 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid (E-5166). Incidence of skin papilloma, chemically induced in mice, was significantly influenced by dietary vitamin A contents. When given orally at a dose of 200 mg/kg body weight, beta-carotene regressed the skin papilloma to some extent (-16% at 14 days), although its effect was much weaker than that of E-5166 (-43%). E-5166 also significantly reduced tumor incidences of experimental hepatomas induced by chemical carcinogen in rats as well as in "spontaneous" hepatoma-bearing mice (C3H/HeNCrj) genetically determined. Further chemical studies revealed that retinol was locally deficient in the hepatomas but not in adjacent normal livers: In particular, anhydroretinol was newly detected in the tumors of spontaneous hepatoma-bearing mice, suggesting increased conversion of retinol into the inactive metabolite. Moreover, cellular retinoid-binding protein, F-type (an oncofetal protein), also newly appeared exclusively in the hepatoma tissues, suggesting that the preventive effect of E-5166 on hepatocarcinogenesis was mediated, at least in part, through its binding with the new retinoid receptor.

Glutathione and gamma glutamyl transpeptidase in rat liver during chemical carcinogenesis.

Continued administration of several hepatocarcinogens led to an increase in the concentration of glutathione (GSH) in the livers of intact, but not of hypophysectomized or adrenalectomized rats. The concentration of GSH remained high untill the development of hyperplastic nodules. Subsequently, the concentration of GSH dropped to the normal level or below. A single dose of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) produced an increase of GSH which, within a certain range, depended upon the amount of the carcinogen. In well differentiated, slowly growing hepatomas, the concentration of GSH approached the level in normal adult rat liver. On the other hand, in nondifferentiated and rapidly growing hepatomas, GSH was only 30-40% of that in normal liver. The activity of gamma-glutamyl transpeptidase (GTase) increased within 24-48 hours after a single large dose of 3'-Me-DAB. Continued feeding of 3'-Me-DAB led to an exponential increase of GTase. During hepatocarcinogenesis, the level of GTase activity corresponded to the degree and size of pathologic changes produced in rat liver. Chloramphenicol partially inhibited the increase of GTase induced by 2-acetylaminofluorene. Pretreatment with 3-methylcholanthrene partially inhibited the increase of GTase that had been induced by a single dose of 3'-Me-DAB. Puromycin partially inhibited the increase of GTase induced by several doses of dimethylnitrosamine. These observations indicated a close connection between the activation of GTase and chemical carcinogenesis in rat liver. Measurements of GTase activity in 12 Morris hepatomas supported this conclusion; their GTase levels were greatly elevated compared with that in normal adult rat liver.

Malignant transformation of rat liver cells by N-benzoyloxy-3'-methyl-4-methylaminoazobenzene and its related compound.

N-Benzoyloxy-3'-methyl-4-methylaminoazobenzene-(N-benzoyloxy-3'-Me-MAB) was highly toxic to rat liver epithelial cells (Ac2F) and induced many more chromosome aberrations and unscheduled DNA synthesis than did 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). N-Benzoyloxy-3'-Me-MAB and its related compound, N-acetoxy-4-methylaminoazobenzene, efficiently induced malignant transformation of the cells, but 3'-Me-DAB was not so effective. The cells transformed by N-acyloxy-4-methylaminoazobenzene (N-acyloxy-MAB) derivatives showed significant increases in plating efficiency in liquid medium and in the size of aggregates in rotation culture. Such increases were not seen in the 3'-Me-DAB-transformed cells. The results indicate that the N-acyloxy form of the 4-dimethylaminobenzene derivatives seems very likely to be the ultimate metabolite.

Decrease in L-type pyruvate kinase activity in rat liver by some promoters of hepatocarcinogenesis.

A probably promoter-specific decrease of L-type pyruvate kinase (L-PK) in Wistar rat liver is described. The possibility of utilizing the decrease in L-PK activity for screening of hepatic promoters is discussed. A significantly decreased level of activity of L-PK was observed during continuous feedings of the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene [(3'-MeDAB) CAS: 55-80-1], which initiates and promotes hepatocarcinogenesis, and of the known hepatic promoters phenobarbital [(PB) CAS: 50-06-6] and dichlorodiphenyltrichloroethane (CAS: 50-29-3) for at least 4 weeks. In contrast, if it occurred, the decrease in L-PK activity by the nonpromoting agents amobarbital (CAS: 57-43-2) and diphenylhydantoin. (CAS: 57-41-0) was temporary and almost overcome by the 4th week. The depression of L-PK activity caused by PB was reversible, was inversely correlated with PB concentration in the diet, and seemed to be organ-specific. Although hepatic promoters lowered L-PK activity in this study, data are so limited that a much more extensive study is necessary before a general conclusion can be drawn. In contrast to L-PK activity, the activity of K-type pyruvate kinase (K-PK) was induced by injections of the carcinogen diethylnitrosamine (CAS: 55-18-5) or the hepatotoxin CCl4 (CAS: 56-23-5) or by the feeding of 3'-MeDAB. However, feeding of PB or 2-methyl-4-dimethylaminoazobenzene (CAS: 54-88-6), which initiates but does not promote hepatocarcinogenesis, did not increase K-PK activity.

Changes in th circadian rhythm of ornithine decarboxylase in rat liver during chemical hepatocarcinogenesis.

The chronobiology of ornithine decarboxylase (ODC) activity in livers was investigated in noninbred Sprague-Dawley rats fed for 5 months with a basal diet or diets with 3-methyl-4'-(dimethylamino)azobenzene (3-Me-DAB) that were oncogenic or caused bile duct hyperplasia (1-naphthylisothiocyanate) (NIT). After a transient disappearance of the ODC circadian rhythm during month 1 on the oncogenic diet, this rhythm in the livers of the rats was reestablished at 60 and 90 days and then disappeared for the next 2 months. When present, the ODC rhythm in 3-Me-DAB-treated rats had the same daily temporal pattern as that of the controls. In the livers of rats treated with NIT, the ODC circadian rhythm was never detectable, even after only 1 month of feeding. Generally, the 3-Me-DAB feeding induced higher levels of ODC activity than did the NIT feeding. The alternation of the appearance and the disappearance of ODC circadian rhythm might reflect changes in the cell population during neoplastic transformation. Te chronobiologic differences in ODC rhythm between the group fed 3-Me-DAB and the group fed NIT could be related to the different types of proliferating cells involved in the hepatic responses to the two drugs.

Enhancement of hepatocarcinogenesis by sorbitan fatty acid ester, a liver pyruvate kinase activity-reducing substance.

Effect on hepatocarcinogenesis of dietary sorbitan fatty acid ester (SorFAE), which had been known to cause decrease in pyruvate kinase (PK) activity, was studied in rats fed a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for 6 weeks. The incidence of hyperplastic nodules and/or hepatocellular carcinomas in the rats fed the 3'-Me-DAB diet alone was 45.0% at the end of 51 weeks, whereas the incidence in the rats fed 3'-Me-DAB diet followed by 5 or 10% SorFAE or 0.1% phenobarbital (PB) diet were 76.2, 90.5, and 95.0%, respectively. These incidences were significantly higher compared with the group fed 3'-Me-DAB diet alone (P less than .05). No tumors were observed in rats fed 10% SorFAE diet alone. The results show that SorFAE has an enhancing effect on hepatocarcinogenesis, although the effect was weak compared to that of the effective PB dose. The results seem to confirm our assumption that a chemical that causes decrease in PK activity in rat liver might promote hepatocarcinogenesis.

Modifications of 3'-methyl-4-dimethylaminoazobenzene carcinogenesis of rat liver and carcinogen metabolism by portacaval anastomosis.

The effect of portacaval shunt on hepatocarcinogenesis was studied in rats fed 3'-methyl-4-dimethylaminoazobenzene. Portacaval anastomosis resulted in a decrease of hepatocarcinogenesis as reflected by a delay in the early peak of alpha-fetoproteins, an absence of late appearance of alpha-fetoproteins, and a significantly lower incidence of tumors than in nonshunted rats. Reduction of hepatocarcinogenesis in shunted rats was associated with a decrease of the binding of 3'-methy-4-dimethylamioazobenzene metabolites to liver proteins. This effect seemed to be related to modifications of carcinogen-metabolic pathways. While the detoxifying azoreductase activity was not affected by portal diversion, the activating pathway leading to the binding of 4-dimethylaminoazobenzene metabolites to DNA, a major step for cell carcinogenesis that is mediated by microsomal enzymes, was decreased in shunted rats to about 50 percent of control values. The decrease of liver weight that occurred in shunted rats without loss of body weight produced a very significant reduction of the total capacity of liver to activate 4-dimethylaminoazobenzene while the total capacity of detoxification remained unchanged. This could be a direct consequence of portacaval anastomosis, as has been shown for other microsomal enzymes.

A phenylalanine transfer RNA azo dye complex in livers of rats fed diets containing 3'-methyl-4-dimethylaminoazobenzene.

A highly purified phenylalanine transfer RNA (tRNAPhe) was isolated from a normal rat liver and from livers of male and female rats fed a semisynthetic diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene for 3 weeks. Absorption spectral analysis on tRNAPhe from normal and carcinogen-fed rats indicated an unusual absorption above 335 nm by tRNAPhe isolated from the latter group. Ribonuclease T1 digestion followed by chromatography on diethylaminoethyl cellulose columns indicated the existence of a major peak of covalently bound oligonucleotide-azo complex. Microcrystalline cellulose thin-layer chromatography resolved the peak into one major and four minor components, all with similar absorption spectra. Enzymatic digestion of the major peak obtained from diethylaminoethyl cellulose chromatography to ribonucleosides, followed by chromatography on cellulose thin layer, resulted in one major and three minor components. A higher uridine:cytidine base ratio was also observed in the tRNAPhe isolated from the carcinogenfed animals. These findings suggest that certain transfer RNA's may be major targets for this azo carcinogen.

Binding of 3'-methyl-N,N-dimethyl-4-aminoazobenzene metabolites to rat liver cytosol proteins and ligandin subunits.

Twenty min after i.p. administration of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene in corn oil to rats, 0.73% of administered radioactivity was present in the liver. Only 0.45% of radioactivity present in liver was recovered in the nuclear fraction, whereas 25% was present in the cytosol fraction. Twenty-seven % of cytosolic radioactivity was trichloroacetic acid precipitable, and 2% was immunoprecipitable with monospecific anti-rat liver ligandin immunoglobulin G. After 3 hr of administration, 3.2% of administered radioactivity was present in the liver, 40% of which was in the cytosol. Although 59% of radioactivity present in liver cytosol was trichloroacetic acid precipitable as compared to 27% at 20 min, the radioactivity precipitated by anti-ligandin immunoglobulin G was still 2%. When liver cytosol obtained from rats after 20 min of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene administration was fractionated on a Sephadex G-75 column, three peaks of radioactivity were observed. When cytosol was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was mainly associated with 5 proteins with molecular weights of 88,000, 47,000, 41,000, 31,000, and 22,000. When the immunoprecipitate obtained from cytosol with anti-ligandin immunoglobulin G was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was exclusively associated with the subunit of ligandin with a molecular weight of 22,000. Approximately 90% of the radioactivity in the immunoprecipitate was covalently associated with this subunit. These studies reveal that 3'-methyl-N,N-dimethyl-4-aminoazobenzene or its metabolites are selectively bound to the subunit of ligandin with a molecular weight of 22,000 and four other cytosol proteins in vivo.

Promotion by dietary phenobarbital of hepatocarcinogenesis by 2-methyl-N,N-dimethyl-4-aminoazobenzene in the rat.

The hepatocarcinogenicity of 2-methyl-N,N-dimethyl-4-aminoazobenzene, previously shown to be noncarcinogenic in adult rats in the absence of further treatment, was observed by following a 1- to 6-week period of feeding this dye to weanling rats with the dietary administration of 0.05% phenobarbital for up to 70 weeks. Many large hepatocellular carcinomas developed in the phenobarbital-treated animals by 72 weeks, whereas a very small number of tiny neoplastic nodules, including one carcinoma, were seen in the rats not given this drug. This study suggests that the use of promoting agents, following the short-term administration of weak carcinogens for the liver, can be useful in demonstrating the initiating activity of such compounds. This system may be useful in the identification of such agents in the environment.