Investigations into the hepatic metabolism of dimethylnitrosamine in the rat.

In this paper we report the detection and identification of methanol as an intermediate formed during both the $\text{in vivo}$ and the $\text{in vitro}$ metabolism of dimethylnitrosamine (DMN) in the rat. Methanol was formed in both hepatic 10,000 $\text{g}$ av. supernatant and washed microsomal fractions over a wide range of nitrosamine substrate concentrations. Furthermore the total amounts of methanol and formaldehyde formed largely accounted for the metabolic fate of both methyl moieties of DMN. Although a number of inhibitors of alcohol metabolism profoundly inhibited the hepatic metabolism of DMN they had little effect on the activities of two mixed function oxidase dependent enzymes. The results suggest that DMN and possibly other dialkylnitrosamines are degraded by enzymic pathway(s) not dependent on cytochrome P-450.     

Effect of calcium phosphate and alumina C γ gels on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system

When CaCl₂ was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately $\text{built1}\text{3}$ of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining $\text{2}\text{3}$ of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C γ gels could substitute for CaCl₂ in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C γ gel can exert its effect in the absence of both CaCl₂ and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.     

Lack of mutagenicity and metabolic inactivation of aphidicolin by rat liver microsomes

In view of the possible utilization of aphidicolin, a specific inhibitor of DNA polymerase α, in the treatment of neoplastic diseases, it seemed important to assess the mutagenic effect of the drug and the possible modification induced by metabolic activation in the liver. This paper shows that aphidicolin lacks mutagenicity in the Ames' $\text{Salmonella}$-microsome test in agreement with our previous observation that it does not induce DNA repair synthesis in HeLa cells. During the studies of mutagenicity we have observed that aphidicolin is converted to inactive derivative(s) by rat liver microsomal oxidases. The reaction is dependent on time and temperature and requires NADP⁺ and glucose-6-P. The metabolites are not mutagenic and they do not induce DNA repair synthesis in HeLa cells. Therefore the possible anti-cancer use of aphidicolin is not hampered by its partial metabolic inactivation in liver. Our results suggest however that aphidicolin will possibly be clinically useful at concentrations higher than those expected from our studies with human DNA polymerase $\text{α}\text{in vitro}$ and human neoplastic cell lines $\text{in vivo}$. The metabolic derivative(s) of aphidicolin is inactive both against cellular DNA polymerase α and Herpes simplex viral DNA polymerase.     

Methylation of DNA in rat liver and intestine by dimethylnitrosamine and N-methylnitrosourea

A chromatographic procedure for improved separation of deoxyribonucleosides and methylated deoxyribonucleosides is described. DNA was isolated from liver and small intestine of rats treated with [¹⁴C]dimethylnitrosamine ([¹⁴C]DMN) or $\text{N-[}{}^3\text{H}\text{]}\text{methyl}\text{-N-}\text{nitrosourea}$ ([³H]MNU), and the purified DNA was hydrolyzed enzymatically. The deoxyribonucleosides were chromatographed on an Aminex A-6 cation exchange column at 37°C with 0.4 M ammonium formate, pH 4.5, as eluant. In addition to showing the presence of the expected alkylated products, N⁷-methyldeoxyguanosine (determined as N⁷-methylguanine) and O⁶-methyldeoxyguanosine, several other minor methylated products were found in liver and intestinal DNA of rats treated with DMN or MNU. Two of these products are believed to be N³-methylthymidine and O⁴-methylthymidine.     

Potentiation of dimethylnitrosamine genotoxicity in rat hepatocytes isolated following ethanol treatment in vivo

Unscheduled DNA synthesis (UDS), following exposure to dimethylnitrosamine (DMN), was potentiated in cultured hepatocytes isolated following treatment of rats for 14 or 28 days with 20% ethanol/5% sucrose solution. Ethanol treatment was associated with increased UDS, a concomitant increase in hepatic microsomal protein concentration and DMN N-demethylase activity. Increased aniline hydroxylase activity of hepatic microsomes from ethanol-treated rats preceded the measured increase in microsomal protein content or DMN metabolism. The increase in metabolism of DMN in vitro and potentiation of DMN-induced UDS associated with ethanol treatment may contribute to a synergistic effect of ethanol on DMN hepatotoxicity and carcinogenicity. In contrast, ethanol pretreatment did not increase the cytotoxicity of DMN as characterized by enzyme release.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Metabolic activation of dimethylnitrosamine and diethyl-nitrosamine to mutagens

Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) are not mutagenic by themselves, but they can be converted by mammalian enzymes to highly mutagenic products. As indicators for mutagenic activity, Neurospora crassa and Salmonella typhimurium were used. The ad-3 forward-mutation system was used to detect specific locus mutations; mutants in this system can range from multi-locus deletions to leaky mutations. The induction of mutations in S. typhimurium is detected as induction of histidine revertants of the histidine-requiring strain G₄6. The activation of DMN is microsomal, inhibited by SKF 525-A, and requires co-factors. The activating enzyme is induced in mice by pretreatment with phenobarbital, 3-methylcholanthrene and butylated hydroxytoluene. The mutagenic activity of the reaction products is directly correlated with the metabolic formation of formaldehyde with and without induction by 3-methylcholanthrene and across strains of mine. Formaldehyde does not contribute to the mutagenic activity of the reaction products. It is clear from the data that the reversion sites in G₄6 are more sensitive than the ad-3 loci of Neurospora crassa to the mutagenic action of DMN metabolites formed by mammalian liver. The microsomal assay is a few orders of magnitude more sensitive than the intraperitoneal host-mediated assay, and the intrahepatic host-mediated assay is a few orders of magnitude more sensitive than the in vitro microsomal system.     

Expression and subcellular distribution of mouse cytochrome P1-450 mRNA as determined by molecular hybridization with cloned P1-450 DNA

Cytochrome P₁-450 (P₁-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P₁-450 structural gene [Negishi $\text{et}\text{al.}\text{,}\text{Proc. Nat. Acad. Sci. U.S.A.}\text{78}$: 800–804 (1981)]. Poly(A⁺)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of $\text{Ah}$-responsive $\text{C57BL}\text{6N}$ (B6) and $\text{Ah}$-nonresponsive $\text{DBA}\text{2N}$ (D2) inbred mice. The poly(A⁺)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [³²P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P₁-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein.Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P₁-450 mRNA preceding the synthesis of apo-P₁-450 and the increase of AHH activity.     

Crude oil effects on microsomal mixed-function oxidase system components in the striped mullet (Mugil cephalus)

The effects of exposure to two types of crude oil on microsomal mixed-function oxidase system components in livers of juvenile striped mullet ($\text{Mugil cephalus}$) were investigated. Mullet were exposed for 4 days to emulsified Empire Mix or Saudi Arabian crude oils at an initial concentration of 75 ppm and an average of 1 ppm in the water column. Liver size was increased by about 50% following exposure to both oils. Since neither total hepatic protein nor microsomal protein increased as rapidly as did liver size, the concentrations of both were reduced following oil exposures. The proportion of microsomal protein to total hepatic protein or wet weight was not altered following crude oil exposure. Both cytochromes P-450 and $\text{b}$₅ were induced following oil treatment. NADPH-dependent enzymes assayed with cytochrome $\text{c}$ and dichlorophenolindophenol as substrates showed increases in activity after exposure to Empire Mix crude oil but only the latter enzyme activity was increased on a microsomal protein basis following Saudi Arabian crude oil treatment. Activities of NADH cytochrome $\text{c}$ and NADH cytochrome $\text{b}$₅ reductases appeared to vary with the protein level. However, since liver size was increased, oil-treated mullet had more of all parameters measured than did control mullet. Although the acute toxicity of Saudi Arabian crude oil to mullet is greater than that of Empire Mix crude oil, Empire Mix crude oil had greater inductive effects on microsomal oxidase components.     

Isolation and characterization of an hydroxykynureninase from homogenates of adult mouse liver

A kynureninase-type enzyme was isolated from adult mouse liver. With kynurenine as the substrate, this enzyme has a K_m of 300 μ$\text{M}$; when the substrate is hydroxykynurenine, the K_m is 6 μ$\text{M}$. We conclude that this enzyme is an hydroxykynureninase. No enzyme which we could characterize as a kynureninase was found in this preparation. This suggests that tryptophan metabolism in the mouse occurs primarily through pathways that use hydroxykynurenine rather than kynurenine. Preliminary studies indicate that the enzyme is inhibited by its reaction product, hydroxyanthranilate, which is an intermediate in the synthesis of NAD. Such control of the hydroxykynureninase reaction may be of physiological importance in regulating the synthesis of NAD and/or in preventing the accumulation of hydroxyanthranilate, a putative carcinogen.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Activation of guanylate cyclase and inhibition of adenylate cyclase by cytotoxic preparations from marine sponges of Haliclona genus.

Of seven marine sponges tested only two, $\text{Haliclona}\text{viridis}$ and $\text{Haliclona}\text{rubens}$, yielded preparations that activated rat heart microsomal guanylate cyclase and exhibited direct hemolytic activity. These two preparations also inhibited basal and fluoride-activated adenylate cyclase in rat heart microsomes and glucagon-stimulated adenylate cyclase in rat liver plasma membranes. Hemolytic activity co-purified with nucleotide cyclase-modulating activity during a standard lipid fractionation procedure. This fraction was cytotoxic to 3T3-4a Swiss mouse fibroblasts.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Inhibition of DNA replicative synthesis and DNA repair synthesis in human and mouse cells in culture by cigarette smoke condensate fractions.

Twelve cigarette smoke condensate fractions were tested for their ability to inhibit replicative DNA synthesis and DNA excision repair synthesis in cultures of human fibroblasts and Swiss mouse embryo cells. None of the fractions showed any specificity for the inhibition of DNA repair and, in general, repair synthesis was less sensitive to inhibition than was replicative synthesis. There was some correlation between the inhibitory action of the various fractions and their activity in bioassays performed in other laboratories, including $\text{in vitro}$ cell transformation and bacterial mutagenicity. In most cases, DNA synthesis in the human cells was more sensitive to inhibition than it was in the mouse cells. The specific compounds in the condensate fractions which are responsible for their activity have not been identified.     

Maintenance of microsomal cytochromes b5 and P-450 in primary cultures of parenchymal liver cells on collagen membranes

In previous reports from various laboratories, the levels of the microsomal cytochromes b₅ and P-450 in hepatocytes in primary culture have been found to be very low and difficult to measure. The studies reported in this paper demonstrate that cytochromes b₅ and P-450 in hepatocytes cultured on floating collagen membranes for periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of liver $\text{in}$$\text{vivo}$. Addition of high levels of hydrocortisone (10^?4M) to the culture medium for periods up to 10 days resulted in further increases in the levels of these cytochromes. Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained.     

Differences between products of binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse skin and in a rat liver microsomal system.

Hydrocarbon-deoxyribonucleoside products from the DNA of mouse skin exposed $\text{in vivo}$ to 7,12-dimethylbenz[a]anthracene are chromatographically the same as the products formed in mouse embryo cell cultures. These products, which are known to arise through the generation of a diol-epoxide in the 1,2,3,4-ring of the hydrocarbon, are chromatographically separable from products that result from reaction of the K-region oxide of this hydrocarbon with DNA. However, when 7,12-dimethylbenz[a]anthracene is bound to DNA in the presence of a microsomal system analogous to those used in various carcinogen detection systems, the hydrocarbon-deoxyribonucleoside products co-chromatograph with the K-region oxide products. Differences in the profiles of metabolites formed in mouse embryo cell cultures and rat liver microsomal systems are consistent with the differences between the DNA-bound products in these two systems.     

Organ-specific DNA damage induced in mice by the organotropic carcinogens 4-nitroquinoline 1-oxide and dimethylnitrosamine.

The extent of DNA fragmentation induced in lung, kidney, and liver of mice injected with the chemical carcinogens 4-nitroquinoline 1-oxide (4NQO), dimethylnitrosamine (DMN) and the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO) was estimated by the alkaline sucrose gradient technique. A floating of minced lung tissue pieces in the alkaline lysing solution on top of the gradients afforded a gentle method of lung DNA extraction. This technique minimized mechanical shearing of lung DNA and permitted comparisons to be made with liver and kidney DNA sedimentation patterns. The extent of DNA damage induced by 4NQO followed the order: lung, kidney, liver, while that induced by DMN followed the order: liver, kidney, lung. The sites of greatest DNA damage appeared to correlate with sites of high levels of DNA repair synthesis and the sites of tumor induction. No DNA damage was induced by the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO).     

Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

The template activity of isolated rat liver nuclei for DNA synthesis assayed with $\text{E.}$$\text{coli}$ DNA polymerase was found to be dependent upon the presence of Ca²⁺ or Mg²⁺ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD $\text{in}$$\text{vitro}$.     

DNA synthesis in a sub-nuclear preparation isolated from Physarum polycephalum

A sub-nuclear preparation capable of substantial levels of DNA synthesis $\text{in}\text{vitro}$ has been obtained from isolated S-phase nuclei of $\text{Physarum}\text{polycephalum}$. Nuclei were disrupted by gentle resuspension in a dextran-free medium followed by immediate addition of dextran to stabilize the liberated replication complex. Synthesis continues for at least 120 min, and appears to occur by a semi-discontinuous mechanism. Little DNA synthesis occurs in preparations obtained from G₂-phase nuclei.     

The modifying effect of sodium ascorbate on DNA damage and repair after N-methyl-N′-nitro-N-nitrosoguanidine treatment in vivo

A biological reducing agent, sodium ascorbate, was used to modify both the damage induced by N-methyl-N′-nitro-N-nitrosoguanidine to mouse gastric mucosal cell DNA and the repair of that damage $\text{in vivo}$. Freshly-mixed carcinogen and sodium ascorbate enhanced DNA fragmentation as measured by shifts in alkaline sucrose gradient sedimentation profiles whereas incubation of the two compounds for a short period resulted in reduced DNA fragmentation. Furthermore, periodic administration of sodium ascorbate following stomach cell DNA damage with carcinogen inhibited DNA repair.