Intracellular distribution of hepatic copper in macular mutant mice

To evaluate the role of lysosomes in copper-mediated hepatocellular injury, copper was administered, sc, to both normal and macular mutant mice at doses of 4.5, 9.0, and 18 mg Cu/kg, and the subcellular distribution of copper has been investigated in the liver of normal and mutant mice 24h after injection. The amount of copper in all fractions of copper-treated mutant mice was markedly lower than those in copper-treated normal mice, with the exception of microsomal fraction. However, there were no distinct differences in the proportion of copper in subcellular fractions between normal and mutant mice.     

A study of the copper-binding proteins in liver and kidney tissue of neonatal normal and mottled mutant mice.

The Cu2+-binding proteins from liver and kidney tissue of 7--8-day-old brindled (Mobr) mice and their normal littermates were compared. (1) Separation over Bio-Gel P-10 showed that the differences in the Cu2+ content of mutant tissues were largely associated with a low-molecular-weight protein fraction (mol.wt. 14 500). (2) Further purification of this low-molecular-weight fraction by anion-exchange chromatography revealed four subfractions. The Cu2+ content of each subfraction reflected the Cu2+ status of the tissue of origin; the Cu2+ contents of the mutant kidney subfractions were elevated and those of the mutant liver were depressed compared with normal. In contrast, the protein contents of the subfractions were less variable and did not reflect the differing Cu2+ contents. (3) Amino acid analysis of the four subfractions from CuCl2-treated mutant and normal animals revealed clos similarities. The proteins showed high glycine, glutamic acid, serine, alanine and lysine contents and a rather variable cysteine content. Differences were apparent in the normal liver subfractions, which showed a higher cysteine content and lower glutamic acid content than did either the mutant liver or normal and mutant kidney subfractions. These observations, together with the recorded presence of aromatic amino acids, indicated that these proteins are not thioneins.     

Decreased and disproportionate T-cell population in adult mice after neonatal exposure to diethylstilbestrol

Female Balb/c mice neonatally treated with diethylstilbestrol show persistent impairment of several immunological parameters. The distribution of different classes of lymphocytes in the spleen has been determined at 6, 12, and 18 weeks of age. DES resulted in a decreased percentage of T cells in spleen while the number of B cells was normal. Utilizing Lyt antisera the T-cell subpopulations were found to be imbalanced with an increase in Lyt 123 cells and a concomitant decrease in Lyt 1 cells. Ovariectomy did not influence the diethylstilbestrol-induced alterations in the T-lymphocyte population.     

A comparison of phenotype and copper distribution in blotchy and brindled mutant mice and in nutritionally copper deficient controls

The murine mottled mutants brindled, Mo br, and blotchy, Mo blo, are valuable animal models for the study of mammalian copper metabolism. In this paper, we present data showing that a nutritionally copper deficient suckling mouse, Cu-, with strong phenotypic similarities to the brindled mutant can be produced by feeding genetically normal dams a copper deficient diet (0.1-0.4 ppm Cu2+) from the day of mating. Comparisons of copper distribution between the Cu- mice and brindled mutants indicate that when a small dose of copper (0.5-0.9 micrograms Cu2+) was administered by intracardiac injection, the copper was abnormally distributed, and that the pattern of tissue distribution was very similar in Cu- mice and brindled mutants 24 h after injection. When, however, a treatment dose (50 micrograms Cu2+) was injected subcutaneously, and tissues assayed 3 d after injection, copper distribution in Cu- mice and brindled mutants was clearly different. Copper deficiency in Cu- suckling mice is entirely derived from maternal effects. Evidence that maternal effects may also influence the survival and phenotype of the brindled and blotchy mutants was obtained by comparing the viability of mutants born to dams carrying mottled mutations on one or both X chromosomes.     

The intravenous distribution of a radiolabeled, potentially useful cluster ion of copper and penicillamine

The 64Cu-radiolabeled mixed valence cluster ion of copper and penicillamine (Cu(I)8Cu(II)6Pen12Cl5-) is easily prepared and purified in a 30 minute procedure. Mice were dosed intravenously with this substance at 0.1 mg/kg, and the distribution of the isotope among organs and tissues was quantified by gamma scintillometry of the isotope's positron annihilation radiation. The largest concentration was found in urine (a 96-fold increase over whole body specific activity at 20 minutes), and this finding is consistent with the compound's inulin-like renal clearance. Isotope levels were also elevated in kidney and liver tissues (2.36-and 3.62-fold, respectively), while all other organs and tissues examined were found to be depleted of the label. A pre-injection of unlabeled cluster at 60 mg/kg effectively blocked radiolabel interaction with liver tissue. This intensely red-violet compound did not stain viable liver cells, and it was not significantly decomposed by liver homogenates over a three hour period. These results suggest cluster interaction at saturable binding sites on liver cell surfaces. The unlabeled cluster shows no indication of toxicity, and the labeled version might have biomedical applications.     

Development of copper deficiency in neonatal mice

Dietary copper (Cu) deficiency was produced in Swiss albino mice to determine the temporal relationship between depletion of Cu and changes in the cardiovascular and nervous system. Dams were placed on a Cu-deficient diet 4 days after parturition. Half the dams were provided with deionized water and their offspring are referred to as Cu-deficient (-Cu). Half the dams were given cupric sulfate in their drinking water (20 microg Cu/mL) and their offspring are referred to as Cu-adequate (+Cu). At 6 weeks of age a sample of the -Cu mice were repleted with CuSO(4). Mice were sampled 1 day after birth and at weekly intervals for 7 weeks. Both +Cu and -Cu mice grew at the same rate: birth weight increased 16-fold at 6 weeks of age. Liver Cu more than doubled between 1 and 7 days of age. At 2 weeks of age -Cu mice were anemic (lower hematocrit and hemoglobin) and had lower liver Cu and plasma ceruloplasmin activity compared to +Cu mice. Liver Fe was not elevated in -Cu mice until 2 weeks after anemia developed. At weaning first signs of altered catecholamine metabolism included elevation of dopamine in both heart and spleen. Norepinephrine concentrations and content, in contrast, were not both lowered in -Cu mice until 5 weeks of age. Heart weight was first elevated in -Cu mice at 6 weeks of age and relative weight (mg/g body wt) at 4 weeks of age. Liver Cu concentration was lower in 1-week repleted mice than in +Cu mice. Anemia preceded the development of cardiac hypertrophy and altered catecholamine levels in -Cu mice.     

Hepatic metallothionein synthesis in neonatal mottled-brindled mutant mice

Mottled-brindled mutant mice did not display the elevated hepatic metallothionein synthesis normally observed in 2- to 6-day-old wild-type mice. This difference between normal and mutant mice was not due to a decreased ability to synthesize metallothionein in the liver, since hepatic metallothionein synthesis was inducible in response to copper, cadmium, zinc, or hydrocortisone administration to neonatal mutant mice. Hydrocortisone treatment resulted in increased metallothionein synthesis in liver of mutant mice but had no ameliorative effect on the mottled-brindled disease.This work was supported by Contract DE AM03 76 SF00012 between the Department of Energy and the Regents of the University of California. JEP is the recipient of a National Research Service Award from the National Institutes of Health.     

The distribution of synaptosomal oxidative enzymes after exposure to deoxycholate

Abstract— The distributions of NADH₂ dehydrogenase, NADH, cytochrome c reductase and cytochrome oxidase have been determined utilizing synaptosomal isolation techniques. Deoxycholate was used to determine compartmentation and/or ‘latency’ of these activities. NADPH, dehydrogenase proved to be a soluble and mitochondrial enzyme and the activity of this enzyme was not appreciably changed by deoxycholate treatment. NADH_g cytochrome c reductase proved to be a mitochondrial enzyme with considerable activity in microsomal fractions. Deoxycholate treatment increased activity in the synaptosomal fraction 8.3-fold. A bimodal activation pattern was observed with synaptosomal and mitochondrial NADH, cyrochrome c reductase upon exposure to increasing concentrations of deoxycholate, with enhancement of activity at 0.25 % (w/v) and 0.50 % (w/v) deoxycholate. The enzyme was stable at concentrations of deoxycholate less than 0.25% (w/v) but was irreversibly inactivated at concentrations higher than 0.25% (w/v). The mechanism of this activation pattern appeared to be a combination of enzyme release and inactivation. Similar results were not observed in liver mitochondria. Cytochrome oxidase, a known mitochondrial marker, exhibited a 17-fold increase in synaptosomal activity with deoxycholate treatment. The synaptosomal cytochrome oxidase activity after deoxycholate treatment approached the activity in the free mitochondrial fraction. The percentage of mitochondrial protein in synaptosomal fractions was estimated to be about 30 per cent from a comparison of the respective total (deoxycholate-treated) activities. On the basis of these data we suggest that the synaptosomal fraction possesses a relatively sizable energy-producing potential which may be of significance in vivo.     

Ovarian reproductive function after exposure to diethylstilbestrol in neonatal life

Inbred and random-bred NMRI mice were treated with diethylstilbestrol (DES, 5 micrograms per day) or vehicle (olive oil) on Days 1-5 after birth. At the age of 8 wk, females were treated with saline or eCG and hCG to induce ovulation. Ova never occurred in the ampulla of the uterine tube of saline-treated, DES-treated females when these mice were not mated. After gonadotropin treatment, ova were found in the ampulla of all olive oil-treated females and in approximately 80% of DES-treated females. The number of ovulated ova was similar in both groups. Twenty percent of gonadotropin-treated, DES-treated females had ova in the ampulla and a vaginal plug after being caged with males but none became pregnant. Ovaries from inbred control or DES-treated females were grafted to the ovarian bursa of control or DES-treated ovariectomized hosts. DES-treated hosts, carrying control or DES-exposed ovaries, never became pregnant. Control females, with control ovaries or DES-exposed ovaries, became pregnant; pregnancy rate and litter size were similar for control mice regardless of whether they were supporting DES-exposed or control ovaries. Oocytes from ovaries exposed neonatally to DES can thus give rise to apparently normal offspring. The results also indicate DES-induced nonovarian disturbances, e.g. tubal and/or endometrial function, both of which are important for fertility. In the grafting experiments, a high mortality rate was found in inbred DES-exposed females caged with males. All deaths were associated with vaginal concrements (vaginal stones) and intestinal complications.     

The frequency and distribution of isolabelling in Chinese hamster chromosomes after exposure to x-rays

2 experiments were carried out, following the same scheme. Chinese hamster cells (line CHEF-125) were cultivated for 4 h in the presence of tritiated thymidine ([³H]TdR) (Expt. 1, 0.5 μCi/ml; Expt. 2, 0.1 μCi/ml). After removal of the isotope, some of the cultures were X-irradiated in stage S-G₂ preceding M₁ (series S-G₂ I), others in stage S-G₂ preceding M₂ (series S-G₂ II).Analysis of the colchicine-C-anaphase of M₂ cells showed that in both experiments the X-rays produced an increase in the isolabelling regions only if given in the S-G₂ stage preceding M₁. On the other hand, the X-rays produced an increase in sister chromatid exchanges (SCEs) in both series of the second experiment. In the first experiment, where the controls had a high frequency of SCEs, no increase in SCEs was recorded in either of the two series treated with X-rays. The frequency of the isolabelling regions, in relation to the total labelling (Expt. 2), was 1.4% in the control series, 2.5% in series S-G₂ I and 1.3% in series S-G₂ II.Analysis of the distribution of the isolabelled regions along the chromosome showed that the regions furthest from the centromere were those most involved in this phenomenon and that, for the most part, the isolabelling was not directly associated with an exchange.The results have been interpreted, in accordance with the theory put forward in a previous work, to mean that SCEs and isolabelling regions are due to different phenomena, the former resulting from an exchange involving the whole chromatid, the latter from subchromatid exchanges probably involving a single polynucleotide strand.     

Characteristics of the androgenization produced in mice by neonatal exposure to alpha-fetoprotein antibodies

Neonatal male and female mice, ages 1-5 days, were injected intracranially with either anti-alpha-fetoprotein (AFP) IgG, steroids, or various control solutions. The mice were autopsied 60-70 days later and the spleen, thymus, liver and gonads were examined by light microscopy. The antibody to AFP produced a gonadal response which was sexual specific. Histological examination of the tissue sections from female mice treated with either anti-AFP IgG or steroids revealed the presence of polyfollicular ovaries lacking in corpora lutea formation. A comparable antibody-induced specific effect on the testes could not be demonstrated; however, steroidal administration induced aspermatogenesis and delayed maturation in parallel studies. In the anti-AFP IgG-treated groups, there appeared a gross neurological lesion which was termed external hydrocephaly. The physiologic responses in the female gonad were found independent of the presence of this anatomical brain lesion, whereas those in the male gonad were found causally related. The immunological specificity of the gonadal response was demonstrated by the use of IgG devoid of anti-AFP IgG and by various IgG control solutions. Thus, exposure to anti-AFP IgG during the critical period of sexual development in the brain appears to mimic steroidal androgenization of the female mouse.     

Overexpression of copper/zinc superoxide dismutase does not prevent neonatal lethality in mutant mice that lack manganese superoxide dismutase

There are two types of intracellular superoxide dismutases: the mitochondrial manganese SOD (MnSOD) and the cytoplasmic copper/zinc SOD (CuZnSOD). Mutant mice that lack MnSOD die shortly after birth because of cardiomyopathy and mitochondrial injury. In order to verify if CuZnSOD could compensate for MnSOD deficiency, a new mutant mouse that overexpresses CuZnSOD but is deficient in MnSOD was generated by crossing MnSOD knockout mice with CuZnSOD transgenic mice. CuZnSOD activity was significantly increased in the blood, brain, liver, and heart of MnSOD knockout, CuZnSOD transgenic mice when compared with nontransgenic mice. However, overexpression of CuZnSOD did not prevent neonatal lethality in mice that lack MnSOD, nor did it prevent oxidative aconitase inactivation, nor did it rescue MnSOD-deficient astrocytes in culture. Based on our findings, which emphasize the strong enzymatic compartmentalization of CuZnSOD and MnSOD, therapeutic antioxidant strategies should consider the final intracellular localization of the antioxidant used, especially when those strategies are directed against mitochondrial diseases.     

A study of copper treatment and tissue copper levels in the murine congenital copper deficiency, mottled.

The injection of copper chloride overcomes the lethality and pigment deficiency in the brindled ($\text{Mo}$^br) mouse mutant but copper levels remain depressed in the liver and brain, and a further accumulation occurs in the kidney. The copper-dependent synthesis of brain noradrenaline returns to normal but the activity of brain cytochrome c oxidase, although increased, remains depressed. Significant changes in tissue copper content of female brindled heterozygotes are reported and in each case, the changes exceed those expected on the basis of X-inactivation. The significance of these results to the development of a satisfactory treatment regime for this disease is discussed.     

Effect of in-utero exposure to diethylstilboestrol on ontogeny of uterine glands in neonatal mice

Pregnant CD-1 mice were injected with diethylstilboestrol (10 micrograms/kg body weight) in 0.1 ml maize oil, or maize oil alone, on Day 16 of gestation. Six experimental and 6 control female progeny were killed daily from birth until Day 7 and uterine tissues were examined by light microscopy. In-utero exposure to diethylstilboestrol resulted in hypertrophy of luminal epithelial cells and premature formation of uterine glands. The initial sign of uterine gland formation was invagination of the uterine surface epithelial cell layer into the underlying connective tissue stroma. A temporal difference occurred between control animals and those exposed to diethylstilboestrol: uterine gland formation first occurred in experimental progeny on Day 4, but not until Day 5 in control progeny. Uterine glands which extended deep into the connective tissue stroma to the myometrium were present in diethylstilboestrol-treated progeny by Day 7, but remained in the superficial endometrial connective tissue stroma in control animals. The results indicate that prenatal exposure of mice to diethylstilboestrol causes uterine epithelial cell hypertrophy at birth and the premature formation of uterine glands during the first week of neonatal uterine development.     

Developmental variation in copper,zinc and metallothionein mRNA in brindled mutant and nutritionally copper deficient mice

The concentrations of copper, zinc and metallothionein-I (MT-I) mRNA were determined in the liver, kidney and brain of the brindled mutant mouse from birth until the time of death. Despite accumulation of copper in the kidney of the mutant, MT-I mRNA concentrations were normal. There was no difference between the MT-I mRNA in the brain of mutant and normal in the first 10 days of life, but after day 10 metallothionein mRNA levels were increased in the mutant. The concentration of copper was very low in the liver of the mutant, and on day 6 after birth the metallothionein mRNA was also reduced by about 50%. This reduction was not seen in copper-deficient 6-day-old pups, despite very low hepatic copper levels. This suggests that the lower hepatic MT-I mRNA in the day 6 brindled mouse was not simply due to the reduction in hepatic copper and also that hepatic copper is not regulating metallothionein gene expression the liver of neonatal mice. After day 12 hepatic MT-I mRNA levels were elevated in mutant and in copper deficient mice, both of which die at 14 to 16 days. These increases and the increase in brain MT-I mRNA in older mutant mice are likely to be caused by stress. Overall the results support the conclusions that the brindled mutation does not cause a constitutive activation of the metallothionein genes, and that the differences in metallothionein mRNA between mutant and normal are most probably secondary consequences of the mutation.     

In utero exposure of mice to diethylstilbestrol alters neonatal ovarian follicle growth and development

The prenatal exposure of mice to diethylstilbestrol (DES, 10 micrograms/kg on day 15 of gestation) caused both quantitative and structural alterations in ovarian follicles within the neonatal ovary. At birth, control ovaries consisted of small type 1 and 2 ovarian follicles located in the ovarian cortex. By postnatal day 7, ovarian follicle development had advanced to the type 4 stage with larger follicles located within the ovarian medulla. In DES-exposed animals, ovarian follicle maturation was advanced with type 3b and 4 follicles appearing 24 h prior to their appearance in control animals. Also, type 5 ovarian follicles were present on postnatal day 6 in experimental animals but were never seen in control animals. In addition to an alteration in ovarian follicle dynamics, the diameter of individual ovarian follicles was transit time between the various stages of follicular development which results in a greater number of developmentally advanced ovarian follicles being present during neonatal ovarian development. The mechanism by which prenatal exposure to DES alters ovarian follicle dynamics during neonatal development is not known.     

贫铀经口慢性染毒对小鼠肠道菌群多样性的影响

目的观察贫铀经口慢性染毒对小鼠肠道菌群多样性的影响。方法通过将不同剂量的贫铀混入饲料中饲养小鼠,以小鼠肝脏和肾脏铀含量作为判断贫铀在动物体内蓄积的指标,建立贫铀经121慢性染毒小鼠模型,观察各组小鼠之间的体重变化,对各组小鼠进行基于细菌16SrRNAV6－V8区的PCR－DGGE分析。结果食入贫铀的各组小鼠肝脏和肾脏铀含量显著高于对照组（P〈0．05）,各组小鼠体重差异无统计学意义（P〉0．05）,各组小鼠V6－V8IXDGGE图谱的丰富度和多样性指数差异均无统计学意义（P〉0．05）。结论贫铀经日慢性染毒对小鼠肠道菌群多样性无显著影响。     

Developmental variation in copper, zinc and metallothionein mRNA in brindled mutant and nutritionally copper deficient mice.

The concentrations of copper, zinc and metallothionein-I (MT-I) mRNA were determined in the liver, kidney and brain of the brindled mutant mouse from birth until the time of death. Despite accumulation of copper in the kidney of the mutant, MT-I mRNA concentrations were normal. There was no difference between the MT-I mRNA in the brain of mutant and normal in the first 10 days of life, but after day 10 metallothionein mRNA levels were increased in the mutant. The concentration of copper was very low in the liver of the mutant, and on day 6 after birth the metallothionein mRNA was also reduced by about 50%. This reduction was not seen in copper-deficient 6-day-old pups, despite very low hepatic copper levels. This suggests that the lower hepatic MT-I mRNA in the day 6 brindled mouse was not simply due to the reduction in hepatic copper and also that hepatic copper is not regulating metallothionein gene expression the liver of neonatal mice. After day 12 hepatic MT-I mRNA levels were elevated in mutant and in copper deficient mice, both of which die at 14 to 16 days. These increases and the increase in brain MT-I mRNA in older mutant mice are likely to be caused by stress. Overall the results support the conclusions that the brindled mutation does not cause a constitutive activation of the metallothionein genes, and that the differences in metallothionein mRNA between mutant and normal are most probably secondary consequences of the mutation.     

Copper metabolism in mottled mouse mutants. The effect of copper therapy on lysyl oxidase activity in brindled (Mobr) mice.

Lysyl oxidase activity in extracts of skin from 1-day-old Mobr/Y mice was found to be between 50 and 60% of that in corresponding extracts from littermate +/Y mice of the same age. It was increased to 84-150% of that in the latter by prior treatment of the Mobr/Y mice at 7 days of age with a single subcutaneous injection of 50 micrograms of copper, retained as Cu+ in an alkyl polyether/sebacic acid solution. This suggests that in this form the copper is able to by-pass the block in copper metabolism and is deliverable to copper-requiring processes.