Action of polychlorinated biphenyls (Kanechlor 500 and Delor 106) and 3-methylcholanthrene on the activity of some rat liver microsomal enzymes]

The influence of 3-methylcholanthrene and two commercially available mixtures of polychlorinated biphenyls (Kanechlor 500 and Delor 106) on the activity of some microsomal rat liver enzymes has been investigated. The studies included the demethylation of ethylmorphine, dimethylnitrosamine, and 4-dimethylaminoazobenzene as well as the investigation of the activity of aryl hydrocarbon hydroxylase and of azoreductase using benzo(a)pyrene and 4-dimethylaminoazobenzene as substrates, respectively. 3-methylcholanthrene induced the aryl hydrocarbon hydroxylase and azoreductase and led to an increase in the demethylation of 4-dimethylaminoazobenzene but not in the ethylmorphine demethylation. The relatively low increase in the dimethylnitrosamine demethylation was not statistically significant. These polychlorinated biphenyls caused a significant increase in all the enzyme activities studied. In most cases Delor 106 was more active than Kanechlor 500. The results are discussed and compared with those of other authors.     

FINE STRUCTURAL ALTERATIONS IN CELL PARTICLES DURING CHEMICAL CARCINOGENESIS : III. Selective Action of Hepatic Carcinogens Other than 3'-Methyl-4-Dimethylaminoazobenzene on Different Types of Mitochondrial Swelling. Effect of Stimulated Liver Growth

The previous studies on the correlation between tumor incidence and changes in microsomal and mitochondrial swelling during feeding of 3'-methyl-4-dimethylaminoazobenzene to rats have been extended to other hepatic carcinogens. Administration of 4'-fluoro-4-dimethylaminoazobenzene, 4'-ethyl-2-methyl-4-dimethylaminoazobenzene, 2-acetylam-inofluorene, ethionine, and tannic acid were found to produce drastic alterations of the swelling of rat liver mitochondria. In contrast to these compounds, feeding of the non-carcinogenic azobenzene and 4-diethylaminoazobenzene produced only small changes in swelling. Significant modification in the over-all pattern of the swelling curve was observed when the usual concentration of 3'-methyl-4-dimethylaminoazobenzene was reduced, but not when the riboflavin level in the diet was increased tenfold. Feeding of high levels of this dye to the guinea pig did not affect mitochondrial swelling which is consistent with the resistance of this species to azo-dye carcinogenesis. Hypophysectomy provides protection against the alterations, produced by feeding 3'-methyl-4-dimethylaminoazobenzene, in the characteristics of thyroxine- or mercuric chloride-induced mitochondrial swelling. Studies with citric cycle substrates on mitochondrial swelling suggest block of the glutamate α-keto-glutarate pathway after feeding 3'-methyl-4-dimethylaminoazobenzene for 4 weeks. There is a considerable, but reversible, reduction of certain types of mitochondrial swelling in two situations associated with rapid liver growth: after partial hepatectomy and after intraperitoneal injection of 20-methylcholanthrene. Naphthacene, however, which also stimulates rapid liver growth, does not affect mitochondrial swelling.     

Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some uncharged molecules (2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone) that bind to ligandin and aminoazo-dye-binding protein A

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we have examined the interactions of 2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylcholine/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. At 25 degrees C and pH7.4, the partition coefficients for binding to phosphatidylcholine [expressed as (mol of ligand bound/mol of phosphatidylcholine)/unbound ligand concentration] were: for 2-acetylaminofluorene, 5.0x10(3) litre.mol(-1); for 4-dimethylaminoazobenzene, 2.1x10(4) litre.mol(-1); for oestrone, 3.1x10(3) litre.mol(-1); and for testosterone, 4.2x10(2) litre.mol(-1). In the ranges studied these values were independent of concentration. The results for the two steroids confirm those of Heap, Symons & Watkins [(1970) Biochim. Biophys. Acta218, 482-495]. 3. The introduction of cholesterol into the lipid bilayers caused large decreases in the partition coefficients of oestrone and testosterone, but had relatively little effect on the binding of 2-acetylaminofluorene and 4-dimethylaminoazobenzene. 4. By assuming that the interactions with egg phosphatidylcholine bilayers resemble those with the phospholipid components of mammalian intracellular membranes the phosphatidylcholine partition coefficients, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 98, 99, 89 and 58% of the total 2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone respectively may be membrane-bound.     

The possible role of azoreduction in the bacterial mutagenicity of 4-dimethylaminoazobenzene (DAB) and 2 of its analogues (6BT and 5I)

The extent to which azoreductive cleavage contributes to the bacterial mutagenicity of 3 azo compounds has been investigated. The compounds studied were the rodent-liver carcinogens 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT), and the reported non-carcinogenic isostere 5-dimethylaminophenylazoindoline (5I). Although each of these compounds is mutagenic to Salmonella when evaluated using a pre-incubation protocol and in the presence of an induced rat-liver S9 mix, the constituent amines (cleavage products) were essentially inactive. It is therefore concluded that the mutagenic response reported for DAB, 6BT and 5I is related to metabolic activation of the intact molecules. In addition, the non-mutagenicity of 4'-phenyl-4-dimethylaminoazobenzene (4PhDAB) suggests that azoreductase activity is low in the Salmonella preincubation assay, at least as conducted in this laboratory. In the case of 4PhDAB, less than 1.4% azoreduction would yield sufficient quantities of the derived amine, 4-aminobiphenyl, for a positive mutagenic response to have been observed.     

Effect of 3'-methyl-4-dimethylaminoazobenzene in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations in a diploid clone derived from normal rat liver cells in culture

The effect of 3' methyl-4-dimethylaminoazobenzene (3'-Me-DAB) in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations was studied in a diploid strain derived from normal rat liver cells. The cells were malignantly transformed by treatment with 3'-Me-DAB 1.7 micrograms/ml for 130 to 221 d or 1.7 micrograms/ml for 53 d followed by 24.9 micrograms/ml for 27 to 77 d. The untreated control cells did not transform spontaneously until the 232nd d in culture. Some properties of the 3'-Me-DAB-treated cells were compared to those of untreated control cells but no reliable marker for predicting the tumorigenic potential of the cells was found. The single addition of 3'-Me-DAB caused little induction of 8-azaguanine-resistant mutations and chromosomal aberrations to the cells. However, mutations and chromosomal aberrations were significantly induced by N-acetoxy-4-methylaminoazobenzene, an active metabolite of 4-dimethylaminoazobenzene or 3'-Me-DAB in the presence of liver microsomes.     

Change in the mixed function oxidase system of liver microsomes in rats treated with 3'-methyl-4-dimethylaminoazobenzene.

The mixed function oxidase systems of plasma membranes, the Golgi apparatus, and smooth and rough endoplasmic reticula of the livers of rats fed on a standard diet containing 0.06% (w/w) 3'-methyl-4-dimethylaminoazobenzene were investigated. The components and activities of the mixed function oxidase systems of the smooth endoplasmic reticulum and Golgi apparatus were especially reduced by the carcinogen. The activities for hydroxylation of anilines and demethylation of p-nitroanisole and the contents of cytochromes P-450 and b5 of the submicrosomal fractions of the rats decreased considerably more than the activities of NADH- and NADPH-ferricyanide reductases. More than 90% of the ferri-cytochrome P-450 content of the 3'-methyl-4-dimethylaminoazobenzene induced microsomes at 20 K was in the low spin form.     

Proliferation of poorly differentiated cells in the rat liver after a single injection of the hepatocarcinogen 4-dimethylaminoazobenzine and its noncarcinogenic analog

With the aid of autoradiography, the proliferation of non-differentiated (oval and transitional) cells in rat liver induced by a single injection of hepatocarcinogen 4-dimethylaminoazobenzene and its non-carcinogenic analogue has been studied. These cells are characterized by a high proliferative activity reaching its maximum 4 days after injection of both the agents. Later on, the proliferative activity decreased due probably to differentiation of such cells.     

The expression of alpha-fetoprotein and albumin genes in rat liver during chemical carcinogenesis

The effect of the hepatocarcinogen 3′-methyl-4-dimethylaminoazobenzene on α-fetoprotein (AFP) and albumin gene expression in rat liver was studied. Serum concentrations of AFP and albumin were measured. Amounts of AFP mRNA and albumin mRNA in rat livers were determined by hybridization of total cytoplasmic RNAs to their cDNAs. Dramatic increases in serum AFP concentrations coincided with increases in AFP biosynthesis and amount of AFP mRNA in livers of carcinogen-treated rats. In contrast, no or little change in albumin mRNA concentration was found in livers of rats treated with 3′-methyl-4-dimethylaminoazobenzene. Concomitantly, there was little change in liver albumin biosynthesis or serum albumin concentrations during hepatocarcinogenesis.     

Alterations in turnover of labelled precursors incorporated into rat liver nuclear macromolecules during azo dye feeding

Continual feeding of either 4-dimethylaminoazobenzene (DAB) or 2-methyl-4-dimethylaminoazobenzene (2-MeDAB) to rats resulted in an increase in the uptake but a decrease in the turnover of [³H]lysine in all the nuclear proteins of rat liver. The pattern of lysine turnover in acidic nuclear proteins from DAB-fed animals was more similar to that of normal acidic nuclear proteins than that from the 2-MeDAB-fed animals. The histone fractions showed an increase in uptake after dye feeding which was greater in the lysine-rich fractions than in the arginine-rich fractions. During DAB feeding both the uptake and rate of turnover of [³H]thymidine were greatly increased, but with the noncarcinogenic 2-MeDAB the uptake of the precursor was lower and the rate of turnover slower than in normal animals. These differences in metabolism in response to azo dye feeding are discussed in relation to azo dye carcinogenesis.     

Antigenic stimulation induces recombination activating gene 1 and terminal deoxynucleotidyl transferase expression in a murine T-cell hybridoma

Secondary rearrangements of the T cell receptor (TCR) represent a genetic correction mechanism which changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. Murine T-cell hybridoma A1.1 was employed to investigate whether antigenic stimulation induced re-expression of recombinase genes and altered TCR Vβ expression. Following repeated antigenic stimulation, A1.1 cells were induced to re-express recombination activating gene (RAG)1 and terminal deoxynucleotidyl transferase (TdT) which are generally considered prerequisite to TCR gene rearrangement. Accompanied with the significant changes in TCR mRNA levels over time, it is suggested that secondary rearrangements may be induced in A1.1 cells, which represent a mature T cell clone capable of re-expressing RAG genes and possesses the prerequisite for secondary V(D)J rearrangement.     

Morphometric and cytochemical characteristics of the hepatocytes isolated from the liver of rats after a single exposure to 4,dimethylaminoazobenzene

Hepatocytes have been characterized that were isolated with sodium perchlorate from the livers of rats both intact and given a single injection of a carcinogen--4-dimethylaminoazobenzene. A decreased number of big hepatocytes (600-900 mkm2) and the appearance of small hepatocytes (75-120 mkm2) were observed 7 days after the carcinogen injection. An excessive accumulation of glycogen was shown in certain hepatocytes. By the 30th day the picture was nearly normal.     

Fine Structural Alterations in Cell Particles During Chemical Carcinogenesis : II. Further Evidence for Their Involvement in the Mechanism of Carcinogenesis. The Swelling of Rat Liver Mitochondria During Feeding of Amino Azo Dyes

Swelling under carefully controlled conditions has been used to study alterations in the structure of rat liver mitochondria as a result of feeding azo dyes. The changes of the swelling properties of the mitochondria during feeding of the hepatocarcinogenic 3'-methyl-4-dimethylaminoazobenzene are essentially comparable to those observed previously with the microsomes, under the same dietary conditions. These alterations in mitochondrial swelling are not related to changes in the amount of these cell particulates per unit weight of tissue, during feeding of this azo dye. As with the microsomes, feeding of the isomeric but relatively noncarcinogenic 2-methyl-4-dimethylaminoazobenzene does not affect swelling. The structural differences between liver and hepatoma mitochondria show up not only in the rate and extent of swelling but also in the form of the curves of pH dependence. The influence of ketones and sulfhydryl compounds on the swelling of normal liver mitochondria were studied, with particular emphasis to the role of sulfhydryl groups in membrane permeability. The sudden steep rise in the tumor incidence in groups of rats fed 3'-methyl-4-dimethylaminoazobenzene for increasing intervals of time occurs at about 4 weeks. This time correlates with the point of the minimum swelling of microsomes and mitochondria isolated from the livers of rats fed this same dye. Thus, a correlation is established between the alterations of the swelling properties of these particulates and the carcinogenic process.     

Detection of cells resistant to the cytotoxic action of CCl4 in the hepatocyte populations of rats following a single exposure to 4-dimethylaminoazobenzene combined with subsequent injections of the tumor promoter phenobarbital

It is found that hepatic cells of intact rats measuring 129-192 mcm2 are resistant to cytotoxical action of carbon tetrachloride (CCl4). After a single interperitoneal injection of the carcinogen 4-dimethylaminoazobenzene, small hepatocytes (64-128 mcm2) appear to be maintained for one month following five injections of phenobarbital. These small hepatocytes are resistant to cytotoxical action of CCl4. The resistance was studied using a cytochemical test on succinate dehydrogenase. A direct dependence exists between the cell size and the sensitivity to CCl4 among large sized hepatocytes.     

Principles of selective inactivation of the virus genome. IV. The effect of UV-irradiation of phage MS2 on its binding with anti-MS2-immunoglobulins

Ultraviolet (254 nm) irradiation of the bacteriophage MS2 results in the decrease of the number of antigenic determinants exposed on the virion surface. The cross-section of the decrease, as measured by the number of anti-MS2 IgG molecules bound per virion, is 10(-16) mm2 per photon. The decrease of the phage-antibody binding proceeds after irradiation with a rate constant of about 5 x 10(-3) min-1. Since the antigenic determinants of the phage MS2 coat protein does not contain photoreactive amino acid residues, the irradiation-induced decrease of the phage antibody binding is determined, most probably, by the shielding of the antigenic determinants. Such shielding could be caused by rearrangement of coat protein molecules and/or of the capsid induced by photomodification of non-antigenic fragments of coat protein and/or of intraphage RNA.     

Localization of embryonic aldolase isoenzyme in rat liver cells after a single injection of the carcinogen, 4-dimethylaminoazobenzene, and its noncarcinogenic analog

The localization of a fetal isoenzyme of aldolase (A) in rat liver cells early after a single injection of carcinogen 4-dimethylaminoazobenzene and its noncarcinogenic analog 4-diethylaminoazobenzene has been studied using the immunofluorescent method. Aldolase A was found in the cytoplasm of oval and "transition" cells. These cells appeared in rat liver as a result of treatment with carcinogen and its analog. In mature hepatocytes aldolase A was not found either in intact rat liver, after the treatment with carcinogen or its analog.     

CSM murray award lecture - functional studies of the Lyme disease spirochete - from molecules to mice

Lyme borreliosis, also known as Lyme disease, is now the most common vector transmitted disease in the northern hemisphere. It is caused by the spirochete Borrelia burgdorferi and related species. In addition to their clinical importance, these organisms are fascinating to study because of the wide variety of unusual features they possess. Ongoing work in the laboratory in several areas will be described. (1) The segmented genomes contain up to two dozen genetic elements, the majority of which are linear with covalently closed hairpin ends. These linear DNAs also display a very high degree of ongoing genetic rearrangement. Mechanisms for these processes will be described. (2) Persistent infection by Borrelia species requires antigenic variation through a complex DNA rearrangement process at the vlsE locus on the linear plasmid lp28-1. Novel features of this recombination process will be presented. (3) Evidence for a new global regulatory pathway of B. burgdorferi gene expression that is required for pathogenicity will be described. The DEAH box RNA helicase HrpA is involved in this pathway, which may be relevant in other bacteria. (4) The mechanism of B. burgdorferi to effectively disseminate throughout its host is being studied in real time by high resolution intravital imaging in live mice. Recent work will be presented.     

Synthesis of alpha-fetoprotein in rat liver after a single injection of the liver carcinogen 4-dimethylaminoazobenzene and its noncarcinogenic isomer 4-diethylaminoazobenzene

Single injection of the hepatospecific carcinogen 4-dimethylaminobenzene and its noncarcinogenic isomer 4-dimethylaminoazobenzene induced the foci of oval and transitional cells in the rat liver. Part of the cells synthesized alpha-fetoprotein. It is suggested that induction of the foci of the above cells and alpha-fetoprotein synthesis are likely to be due to the toxic effect of the two carcinogenic agents.     

Detection of cells resistant to the cytotoxic action of CCl4 in rat hepatocyte populations following a single exposure to diethylnitrosamine and subsequent injections of phenobarbital

A single intraperitoneal injection of hepatocarcinogen diethylnitrosamine induced emergence of a new subpopulation of "small" hepatocytes (64-128 mkm2), disappearing by the 30th day after carcinogen injection. But 5 injections of the tumor promotor phenobarbital 7 days after carcinogen treatment prolonged the existence of such "small" hepatocytes up to 3 months. The quantitative cytochemical measurement of succinic dehydrogenase activity (respiratory enzyme of mitochondria) showed these cells to be resistant to cytotoxic action of CCl4. These data are consistent with the earlier reported results obtained in analogous experiments with 4-dimethylaminoazobenzene and phenobarbital.     

Decreased viscosity of rat-liver DNA treated by 3'-methyl-4-dimethylaminoazobenzene, detected with a new viscometric approach

DNA damage induced in vivo by 3'-methyl-4-dimethylaminoazobenzene (3'CH3DAB) was investigated with 2 differently sensitive techniques: the alkaline elution assay and the viscometric measurement of DNA damage. 3'CH3DAB appeared to be falsely negative with the alkaline elution assay, whereas with the viscometric approach, which is about 30-50 times more sensitive, it appeared positive, and the DNA damage was dose-dependent.     

Transglutaminase activity during the differentiation of macrophages

For the examination of the participation of the microsomal electron transport system in mutagenic activation by 4-dimethylaminoazobenzene (DAB), 4-methylaminoazobenzene (MAB) and their 3′-methyl-derivatives (3′-methyl-DAB and 3′-methyl-MAB), monospecific antibodies to NADPH-cytochrome P-450 reductase, 3-methylcholanthrene-inducible major P-450 (MC-P-448) and phenobarbital-inducible major P-450 (PB-P-450) were used. In Ames' assay system, the antibody to NADPH-cytochrome P-450 reductase inhibited the mutagenicities of DAB, MAB, 3′-methyl-DAB and 3′-methyl-MAB by 94, 94, 90 and 87%, respectively. The antibody to MC-P-448 inhibited their mutagenicities by more than 90%, while the antibody to PB-P-450 inhibited the mutagenicities less than 20%. These results indicate that the microsomal electron transport system, especially MC-P-448, is involved in activation of these dyes.