Adolfo D. Garnica

Cell culture studies of menkes kinky hair disease

The dose response as well as kinetics of uptake and retention of copper and cadmium of normal and Menkes kinky hair disease (MKHD) cultured fibroblasts are described. In basal culture medium, intracellular copper concentration in MKHD fibroblasts was approximately 3 times that of control cultures. The intracellular copper concentration of MKHD cells was significantly higher than that of normal fibroblasts at medium copper concentrations below 20 microgram/ml. Death of MKHD cells occurred at medium copper concentrations between 15 and 20 microgram/ml with an intracellular copper level 3 times that at basal medium. Normal cells died at medium copper concentration above 30 microgram/ml with an intracellular copper concentration 19 times that at basal medium. These observations suggested the existence of a regulatory mechanism for maintenance and control of intracellular copper in normal fibroblasts which is effective at medium copper concentrations below 30 microgram/ml. This system is defective in MKHD fibroblasts. In basal medium MKHD and normal fibroblasts had similar intracellular cadmium concentrations; however, at higher medium cadmium concentrations MKHD cells had increased intracellular cadmium levels. The uptake of both 64Cu and 109Cd was significantly higher in MKHD cells than in normal cells, indicating that the uptake of 64Cu and 109Cd is not impaired in MKHD cells. A higher retention of 64Cu was observed in MKHD cells at both 37 degrees C and 4 degrees C. No obvious trend, however, was observed in the difference of retention of 109Cd between MKHD and normal cells. An impairment of egress of copper in MKHD cells is implicated by these results.

Metal-binding studies of metallothioneins in Menkes kinky hair disease.

Two species of metallothioneins were isolated from both normal and Menkes kinky hair disease (MKHD) patient livers. Atomic absorption determination of metals indicated that the patient liver metallothioneins had lower copper and cadmium content than normals. Isotope exchange studies, carried out by incubating native metallothioneins with copper-64 or cadmium-109 demonstrated a decreased affinity for copper and an increased affinity for cadmium in both MKHD metallothioneins. An hypothesis is proposed in which metallothionein functions as an intracellular copper carrier and is responsible for the transport of copper between the cells and the surrounding. Change in the copper affinity of the metallothioneins was suggested to be the major abnormality in MKHD.

Polyamines in the Developing Mouse Brain

Polyamine levels were determined during pre- and postnatal development of the male and female mouse brain to assess their possible role during normal brain development. Tissue polyamine levels were increased during periods of neurogenesis and increased cell packing density (assessed by DNA levels) in all brain regions: cerebral cortex, cerebellum, hypothalamus, and rhombencephalon-midbrain. Tissue polyamine levels declined subsequently in a tissue-specific manner toward adult levels. In contrast, the polyamine concentrations per cell were decreased during periods of neurogenesis. A later increase in rhombencephalon-midbrain spermidine levels is consistent with the presumed association of this polyamine with myelin. No sex differences were found in these studies.

Inducibility of Metallothionein Biosynthesis in Cultured Normal and Menkes Kinky Hair Disease Fibroblasts: Effects of Copper and Cadmium

Summary: Metallothionein biosynthesis is not induced by extracellular copper in Menkes Kinky hair disease (MKHD) or in normal cultured fibroblasts under the conditions of these experiments. In the presence of copper, MKHD fibroblasts also incorporated less cysteine than did normal fibroblasts. Extracellular cadmium greatly enhanced the uptake of cysteine in both normal and MKHD cultures. By the technique of polyacrylamide gel electro-phoresis, it was demonstrated that metallothionein is induced by cadmium in normal and MKHD-cultured fibroblasts.Speculation: The abnormality in copper homeostasis of MKHD is not caused by an alteration of inducibility of metallothionein. Further investigation of the structure of metallothionein or other copper carrier proteins may uncover the etiology of MKHD.

203 POLYAMINE AND NUCLEIC ACID ALTERATIONS FOLLOWING TERATOGENIC TREATMENT WITH DIPHENYLHYDANTOIN

Polyamines are closely bound to nucleic acids; their levels correlate with nucleic acid synthesis and may decrease during interference with such synthesis. We have previously reported decreases in embryonic polyamines per mg protein after exposure to diphenylhydantoin (DPH) in teratogenic doses. We now report alterations in nucleic acid levels following identical treatment. Primagravid Swiss-Webster mice were injected intra-peritoneally on day 9 with either 88 mg DPH/kg body weight or vehicle and embryos were obtained surgically on day 11 (DPH or control n=13). DNA content was determined spectrophoto-metrically and the polyamines spermidine, spermine, and putrescine were quantitated on a Durrum D-500 amino acid analyzer. There was a 40% decrease in DNA content in the treated embryos (99.4 vs 164 ug DNA). The protein contents decreased 30% compared with controls (2.38 vs 3.38 mg). All experimental polyamines per mg DNA were significantly greater than their controls (P<0.01) (putrescine:0.155±0.034 (std. dev.) vs 0.0834±0.009; spermidine:0.379±0.066 vs 0.264±0.028; spermine:0.177±0.034 vs 0.136±0.026 nM/ug DNA). The DPH-induced polyamine changes are not proportionate to the DNA alterations and thus do not simply reflect a block in DNA synthesis.

STUDIES OF 64Cu AND 109Cd EFFLUX AND UPTAKE IN MENKES KINKY HAIR |[lpar]|MKHS|[rpar]| FIBROBLASTS

Previous studies in our laboratory indicated decreased efflux of Cu and abnormal metallothionein (MT1) in MKHS. To further identify the kinetics of these transport phenomena studies utilizing 64Cu and 109Cd were performed in MKHS and normal fibroblasts. MKHS and normal cells were grown in Cd-containing medium (Cd concentration = 1.5 μg/ml, 109cd concentration 0.15 μg/ml, spec. act. = 17.8 μCi/μg) for 20 hours, chased with non-radioactive medium and analyzed at 0, 5, 10, 20 hours.60Cu efflux studies employed a 20 hour pulse (Cu concentration = 10 μg/ml, 64Cu concentration = 10 μg, sp. act. = 885 μCi/μg Cu) with chase intervals of 0, 5, 12, and 20 hours.These data clearly indicate a pronounced defect in the efflux of Cu in MKHS fibroblasts as compared to normal fibroblasts. The cadmium results suggest some impairment of Cd-release by MKHS fibroblasts. Studies by Goka et al. (Pediatr. Res.10:365, 1976) indicated increased uptake of 64Cu by MKHS fibroblasts. Our studies of 109cd uptake suggest nearly equivalent uptake by MKHS and normal fibroblasts (MKHS/normal = 0.9-1.8). These data imply that the observed apparent increased uptake is not the primary phenomenon.

PLACENTAL COPPER TRANSPORT IN THE BRINDLED MOUSE

The X-linked mouse mutant brindled is a model for Menkes syndrome. In young hemizygotes, reduced liver and brain copper concentrations are associated with neurologic dysfunction. In fetuses copper concentrations in placenta and kidney are higher in brindled than controls while those in liver and carcass are lower. To treat the copper deficiency in brindled young, heterozygotes were injected at 16 or 18 days gestation with copper, 6 mcg/g/dose, as cupric chloride, 18 and 6 hours before sacrifice. Placental, carcass, and hepatic copper concentrations in brindled fetuses increased (p>0.006). Injection of methylprednisolone, 5 mcg/g, 20 hours before the copper, to increase fetal hepatic copper storage through metallothionein induction, resulted only in further increase in the carcass copper concentrations. These data suggest: placental copper transport in brindled fetuses is impaired; hepatic copper binding capacity of control and brindled fetuses is limited and cannot be augmented by pretreatment with methylprednisolone; extra-hepatic copper binding may be increased with methylprednisolone, which implicates induction of extra-hepatic metallothionein in both groups of animals.

IMPAIRED COPPER UTILIZATION IN THE COPPER-DEFICIENT BRINDLED MOUSE MUTANT

The Brindled mouse mutant is a model for the Kinky Hair syndrome. Male Brindled hemizygous suckling mice demonstrate poor growth and neurologic deterioration beginning during the first week of life and ending with death by the end of the second week. Newborn Brindled carriers are asymptomatic with normal brain copper concentration; liver copper concentration is low and the renal copper is high. Newborn carrier pups suckled by Brindled dams put on low copper diets on the day of parturition, however, show deficient weight gain by the age of 6 days and by 12 days weigh approximately one-half their normal litter-mates. Neurologic symptoms developed in the copper-deficient young carriers by age 12-14 days while their normal litter-mates remained asymptomatic. Tissue copper studies demonstrate that the liver and brain copper concentration in the carrier is no different from that of the normals, although the renal copper is greater than twice as high. On copper-deficient diets, the brain and liver concentrations of normal and heterozygous young decrease to comparable levels, but heterozygotes also develop systemic and neurologic symptoms. These observations are thought to implicate an abnormality in copper utilization in Brindled carriers brought out by marginal copper intake.

DEFECTIVE METALLOTHIONEIN IN KINKY HAIR SYNDROME FIBROBLASTS

Menkes Kinky Hair Syndrome (MKHS) is caused by defective copper (Cu) transport. Therapeutic trials with parenteral Cu indicate increased urinary Cu excretion, failure to increase hepatic Cu stores and subnormal ceruloplasmin response. These observations prompted study of Cu transport in cultured MKHS fibroblasts. In basal culture medium the intracellular Cu concentration is 300 ng/mg protein vs. 150 ng/mg in normal fibroblasts. Increasing the Cu concentration of the medium results in cell death at 15-20 μg/ml for MKHS vs. 30 μg/ml for normals. At Cu concentrations in the medium below 20 μg/ml, the Cu content of MKHS cells is twice that of normal cells, while from 30-45 μg/ml the intracellular Cu concentrations of MKHS and normal cells approach unity. These data indicate an inability of MKHS fibroblasts to handle Cu normally at all concentrations. The Cu induction profile of MKHS fibroblasts demonstrates a decrease in cadmium (Cd)-mercaptide absorption at 250 nm in the fraction corresponding to metallothionein (MT), which implies an increased affinity of MT for Cu. In basal medium decreased intracellular Cd levels exist in MKHS fibroblasts. Cd-induction (0.1-1.5 μg/ml) experiments indicate increased uptake by MKHS fibroblasts compared to normals. The ratio of MKHS [Cd]/normal [Cd] (ngm/mg protein or ng/μg DNA) ranged from 1.1-1.9 throughout the Cd-induction range. These results, together with the Zn, Cu, and Cd content of the metallothioneins as determined by atomic absorption spectroscopy, are compatible with an abnormal MT and defective Cu efflux for cultured MKHS fibroblasts.