Effects of carcinogenic and non-carcinogenic chemicals on plasma esterases in BALB/c mice

Esterase profiles of plasma from female BALB/c mice treated with a variety of carcinogenic and weakly- or non-carcinogenic chemicals were analyzed. Mice treated with the potent carcinogens diethylnitrosamine, dinitrosopiperazine, dipropylnitrosamine, dimethylhydrazine, urethane, and dimethyldinitrosopiperazine had similarly altered plasma esterase profiles after 7 days' exposure to the chemicals. The alterations consisted of increased activity in 4 esterase bands. The increased activity persisted in some of the bands after cessation of carcinogen exposure. Exposure to high concentrations of the weakly- or non-carcinogenic compounds nitrosohydroxyproline, nitrosomethoxymethylamine, 1-nitroso-4 methylpiperazine, nitroso-2,6 dimethylpiperidine, and ethyl methanesulfonate caused no obvious plasma esterase alterations. Ingestion of carbon tetrachloride resulted in increased activity in one esterase band with concomitant decrease in a second band. Analysis of serum from test mice for levels of serum glutamic oxaloacetic transaminase, alkaline phosphatase, lactate dehydrogenase-lactate substrate, and D-gamma-glutamyl transpeptidase did not differentiate between mice treated with selected carcinogens and those treated with non-carcinogens and/or carbon tetrachloride.     

Demonstration in vitro of two intracellular inactivators of nitrate reductase from Neurospora

Two different inactivators of nitrate reductase have been found in cell free preparations of Neurospora. T he first (Inactivator I) is very active at pH9, is inhibited by disodium ethylene diamine tetraacetate (EDTA) and is present in all mycelia incubated under all conditions tested; the second (inactivator II) is very active at pH 5, is repressed by ammonia or by a metabilic product of ammonia and derepssed by nitrogen starvation, cannot be derepressed by nitrogen starvation in strain nit-2, in which a number of “ammonia-repressible” enzymes are permanently repressed, and is sensitive to phenyl methyl sulfonyl fluoride.Crude extracts of mycelia contain inhibitors(s) of both inactivators.     

The regulation of the decay of nitrate reductase Evidence for the existence of at least two mechanisms of decay

There seem to be at least two different mechanisms of decay of nitrate reductase in Neurospora in vivo: one which is very sensitive to EDTA and cycloheximide, decreases with mycelial age and is not increased by an increase in temperature from 27 to 37°C, the other which is relatively insensitive to EDTA and cycloheximide, increases with the age of the mycelium and with the above temperature shift.     

Polyamines alter the phosphorylation pattern of chromatin proteins by endogenous protein kinases

The effect of polyamines on the chromatin phosphorylation by endogenous protein kinases was investigated. Polyamines not only selectively stimulated the phosphorylation of chromatin proteins but also concurrently inhibited the phosphorylation of a number of polypeptides. In particular, a 11,000-dalton polypeptide with pI 4.5–5.0 was highly phosphorylated in the absence of polyamines, despite being a minor component whereas the phosphorylation was strongly inhibited in the presence of polyamines.     

Acute biochemical and morphological effects of N-nitrosomorpholine in comparison to dimethyl- and diethylnitrosamine.

Some biochemical and ultrastructural changes induced in the livers of rats treated with N-nitrosomorpholine are described and compared with parallel observations in rats given dimethyl- or diethylnitrosamine. Hepatotoxic doses of the nitrosamines caused inhibition of incorporation of [14C]leucine into hepatic proteins, accompanied by progressive disaggregation of polysomes which paralleled the known time course of metabolism of each compound. Dimethylnitrosamine (DMN) and N-nitrosomorpholine (NM) inhibited incorporation of [14C]orotate into liver RNA but diethylnitrosamine (DEN) caused a slight stimulation of orotate incorporation. Electron microscopy revealed similar hepatic cytoplasmic changed induced by each nitrosamine, including dilation and degranulation of the rough surfaced endoplasmic reticulum and subsequent increase of the smooth endoplasmic reticulum. Nuclear changes differed with each compound, N-nitrosomorpholine having more marked effects than either dialkyl compound. The results are discussed with particular reference to the metabolism of N-nitrosomorpholine in the liver.     

Effect of a single treatment with the alkylating carcinogens dimethynitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. I. Test for induction of liver carcinomas.

A single injection of dimethylnitrosamine (DMN), 12.0-15.6 mg-kg, given to 100 g female rats 24 h after partial hepatectomy, induced hepatocellular carcinoma. No animals receiving DMN without partial hepatectomy developed liver carcinomas. Previous evidence had suggested that the incidence of tumours was highest when DMN was administered during the wave of DNA replication which follows partial hepatectomy. The present experiments made this suggestive evidence statistically significant. A single treatment with diethylnitrosamine (DEN) induced liver cell cancer when given to intact or to partially hepatectomised rats. No tumors developed when another alkylating carcinogen, methyl methanesulphonate (MMS), was administered after partial hepatectomy. The significance of these results in relation to the mechanism of initiation of carcinogenesis is discussed.     

Neurophysiological alterations following fluvalinate administration in mice.

Median lethal dose (LD50) of fluvalinate (Marvik 25EC) was 105 (94.6-116.5 mg/kg, ip) in albino mice. Gross observable signs were dose dependent and indicative of central and peripheral nervous system stimulation. Fluvalinate, at 10.5 and 21.0 mg/kg, ip doses in mice, facilitated maximal electroshock seizures, reduced reaction time in analgesic test and enhanced duration of ether anaesthesia. Acute and subacute (7 days) treatment at lower and higher doses enhanced pentobarbitone sleeping time. Acute and subacute treatment (7 days) with phenobarbitone (50 mg/kg, ip) prior to fluvalinate enhanced toxicity of fluvalinate.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. II. Alkylation of DNA and inhibition of DNA replication.

Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Gastrointestinal microecology of BALB/c nude mice.

The aerobic, facultative, and anaerobic microorganisms cultivable from the stomachs, ilea, ceca, and colons of BALB/c athymic (nu/nu) mice (normal and wasting), thymus-implanted normal nude mice, and their heterozygous (nu/+) littermates were investigated. Ninety-one species representing 23 genera of bacteria and yeasts were isolated from the 27 mice. The wasting nude mice showed significantly lower numbers of lactobacilli in their stomach microbiota than did mice from the other three groups. The littermate animals appeared unique among the four groups in having corynebacteria as a major constituent of their stomach and ileal flora. The normal nude mice appeared to have a more diverse anaerobic stomach flora than their heterozygous littermates. These minor differences are discussed with respect to possible immunological, physiological, and environmental factors as their cause. Because the gastrointestinal microfloras of the mice from the four groups were not radically divergent from each other, it was concluded that loss of T-cell function does not dramatically alter the makeup of the cultivable gastrointestinal microflora in these mice.     

The induction of chromosomal damage in rat hepatocytes and lymphocytes I. Time-dependent changes of the clastogenic effects of diethylnitrosamine,dimethylnitrosamine and ethyl methanesulfonate

Male Wistar rats received a single injection of diethylnitrosamine (DEN), dimethylnitrosamine (DMN) or ethyl methanesulfonate (EMS). After a number of time intervals (up to 56 days) liver cells were assayed for the presence of possible preclastogenic damage by performing partial hepatectomy and subsequent analysis of chromosomal damage (micronucleus formation) in isolated hepatocytes. Peripheral blood lymphocytes from the same animals were collected, stimulated to proliferate and assayed for the frequency of sister-chromatid exchanges (SCEs). Whereas all agents significantly increased frequencies of SCEs in lymphocytes up to at least 28 days (EMS) or 56 days (DMN, DEN) after injection, only the latter 2 compounds gave rise to significantly increased incidences of micronucleated hepatocytes. DMN-induced preclastogenic damage in hepatocytes was lost between 28 and 56 days after injection. After DEN, this type of damage was persistent over the entire experimental period (56 days).When rats treated with DEN did not undergo partial hepatectomy, the frequencies of micronuclei at different time intervals after treatment were at control level. This result, together with those from hepatectomized DEN-treated rats, suggests that it is the persistent character of the preclastogenic damage that is responsible for the occurrence of micronucleated hepatocytes at later time intervals after treatment with DEN, rather than the stability of micronuclei which might eventually have been formed soon after injection.     

Study on the ophthalmic diseases in ICR mice and BALB/c mice.

In pharmaceutical companies and research institutes, many toxicity tests are performed with laboratory animals. This study was performed to produce reference data for eye toxicity tests and to investigate the ophthalmic diseases of 408 ICR mice and 119 BALB/c mice, which are commonly used as subjects in toxicity tests. The experimental animals without clinical disorders were selected regardless of sex. The ophthalmic diseases were examined by using special ophthalmic instruments: direct ophthalmoscope, indirect ophthalmoscope, slit-lamp biomicroscope and focal illuminator. The most prevalent ocular variation within normal limits was hyaloid vessel remnant (ICR mice, 28.2%; BALB/c mice, 31.9%) and the incidence gradually decreased with age. The ocular diseases found in ICR mice were retinal degeneration (9.8%), corneal scar (4.2%), focal cataract (2.2%), anisocoria (1.2%), corneal ulcer (0.2%) and uveitis (0.2%). In BALB/c mice, corneal scar (9.2%), focal cataract (1.7%) and corneal ulcer (0.8%) were the ocular diseases found.     

Norepinephrine and nerve growth factor: Similar proteins phosphorylated in the nuclei of target cells

Treatment of C6 glioma cells with a β-adrenergic agonist in the presence of radioactive phosphate leads to increased radioactivity in two nonhistone nuclear proteins. These proteins are very similar to those in the nuclei of sympathetic neurons whose phosphorylation is stimulated by nerve growth factor.     

Proteolytic activity in bovine milk xanthine oxidase preparations.

Five glycosyl-transferases have been found present in purified hepatocyte nuclei of the rat (mannosyl-, galactosyl-, N-acetyl-glucosaminyl-, N-acetyl-galactosaminyl- and sialyl-transferases); these are capable of fixing specific carbohydrates on to endogenous or exogenous protein acceptors.     

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. III. The carboxyl termini of protein P11 are required for assembly activity

The assembly activity and electrophoretic mobility of a T4 bacteriophage baseplate protein, P11, have been found to be affected by digestion with the proteases trypsin, subtilisin and carboxypeptidase Y. Analysis of the trypsin limit-digestion product of P11 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and size analysis by high performance liquid chromatography indicate that there is a decrease of approximately 5000 in the molecular weight of the P11 molecule or a loss of 2500 in Mr from each of the gp11 subunits of the dimer. During protease treatment P11 demonstrates a time-dependent loss in the ability to interact with the baseplate protein P10 to form the P(10/11) complex, the first assembly intermediate of the T4 baseplate 1/6th arm. Similar treatments of the P(10/11) complex indicate that P11 in the complex is not affected by these proteases. Concomitant with the loss of assembly activity is a change in the electrophoretic mobility of P11 on non-denaturing polyacrylamide gels from a single band to a series of more mobile bands suggesting sequential loss of positive charge. P11 assembly activity is completely lost after removal of the first positive charge. These results suggest that the carboxyl termini of the two gp11 subunits of the P11 molecule are involved in the interaction of P11 with P10 to form the P(10/11) complex. Analysis of the portion of gp11 removed by carboxypeptidase Y demonstrates that there are up to 13 aliphatic and aromatic carboxyl-terminal amino acids.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.