Pediat. Res. 13: 2W205 (1979)

Letter to the Editor: Menkes Metallothionein and Copper Metabolism

OWEN M. RENNERT AND WAI-YEE CHAN

Section of Genetics, Endocrinology and Metabolism and Depanmenls of Pediurrics and Molecular Biologv and Biochemistry, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

We read with great interest uCu Metabolism in Menkes and copper transport deficit of Menkes kinky hair disease cells, par- Normal Skin Fibroblasts by Beratis et al. (1). We were particularly tially purified metallothionein (MT-I and MT-2) from one pleased with this article because it has verifid work that we have Menkes kinky hair disease and two normal livers were analyzed. previously presented on defective effIux of copper in Menkes Menkes kinky hair disease metallothioneins demonstrated reduced kinky hair syndrome fibroblasts (2-4). There are several manu- cadmium, copper, zinc, and sulfhydryl group content. Isotope scripts presently in press (5-7) which present greater detail on the exchange studies, carried out by incubating native metalothi- uptake and efflux processes involved in -CU and radiolabeled oneins with WCu or lWCd, demonstrated decreased afIinity for cadmium transport in fibroblasts from patients with Menkes kinky copper and an increased af3nity for cadmium in both Menks hair disease. kinky hair disease metallothioneins. These reported studies have validated that Menkes kinky hair We believe that these observations implied an abnormality in disease cells contain at least twice the intracellular copper of Menkes kinky hair disease fibroblast copper and cadmium trans- normal fibroblasts at medium copper concentrations below 20 pg/ port which was associated with structural abnormalities in metal- ml. Death of Menkes kinky hair disease fibroblasts occur at lothioneins. We proposed an hypothesis in which metallothionein medium copper concentrations between 15 and 20 pg/ml, well functions as a carrier by combining with cations and carrying below the toxic level for normal fibroblasts. Differences in the them to the site of excretion. intracellular wpper concentration, as well as the cell toxicity We submit these comments in reference to the article by Beratis achieved by increasing medium copper concentrations, suggested et 01. (I) because we believe that our data and theirs are somewhat to us that a regulatory mechanism for intracellular wpper trans- divergent with regard to several points. First, we did not find port was defective in normal fibroblasts when copper concentra- significant radiolabeled copper bound to large molecular weight tions above 30 &rnl were achieved. This phenomenon in normal components in either normal or Menkes kinky hair disease fibro- cells was comparable to that seen in Menkes kinky hair disease blasts. Second, in the speculation of the paper by these authors, fibroblast at all medium wpper concentrations. we disagree with their suggestion that increased binding capacity Intracellular cadmium &ncentrations of Menkes kinky hair of low-molecular weight copper transport proteins (metallothi- disease fibroblasts were similar to that of normal cells (23.6 2 6.4 oneins) would account for the copper transport defect in this ng/mg cell protein vs. 23.5 * 9.3 ng/rng cell protein) in basal disorder. medium, but always were higher at other medium cadmium Additionally, our group previously reported (8) that intracellu- concentrations (0.1- 1.5 @g/ml), indicating an increased cadmium lar copper concentrations in brain are significantly below those of uptake by Menkes kinky hair disease cells. control tissues. Therefore, we would question their statement By using MCu for uptake studies, it was demonstrated that suggesting that copper concentration in whole brain is normal. within the first hour copper uptake was three times as great in The transport studies in our previously cited references have been Menkes kinky hair disease cells as that in normal cells, while that performed at more than the two time points (6 and 24 hr) reported of 'Yd was about the same in both types of cells. Subsequent by Beratis et 411. (l), though the conclusions are basically the same uptake of both radionuclides was higher in Menkes kinky hair the distinctions in eMux do not quite follow the time course disease cells than in normal cells. proposed by these authors. Effluxes of aCu and 'OBCd in both Menkes kinky hair disease Finally, on our experiments cell toxicity was induced in both and normal cells were studied by pulse-labeling techniques with normal and Menkes kinky hair disease fibroblasts at concentra- radionuclides for 20 hr and subsequently chased with nonradio- tions well below 60 dm1of copper. active medium. At both 37 and 4", Menkes kinky hair disease h general we agree with the experimental data reported by cells demonstrated a higher retention of both radionuclides than Beratis et al. (I), but would propose somewhat different postula- did normal cells, indicating an impairment of efflux of both metals tions to explain the phenomenon observed in Menkes kinky hair in Menkes kinky hair disease cells. Lowering the temperature did disease fibroblasts. not increase the percentage of retention of either radionuclide in Thank you for allowing us the opportunity to express these the two types of cells, implying that the efflux process was not opinions. energy requiring. Inducibility of metallothioneins in Menkes kinky hair disease cells by copper and cadmium was investigated utilizing L-(?3]- REFERENCES AND NOTES cystine in the presence of both copper and cadmium. MCufailed, 1. Beratis. N. G., Price. P.. LaBadie. G., and Hirschhorn. K.: -CU metabolism in while 'OBCdsucceeded in inducing metallothionein in both normal Menkes and normal cultulgd skin fibroblasts. Pediair. Res., 12: 699 (1978). and Menkes kinky hair disease fibroblasts. However, the metal- 2. Chan, W. Y., Garnica, A. D..and Rennen 0.M.: Defective metallolhionein in lothionein induced by cadmium in Menkes kinky hair disease kinky hair syndrome fibroblasts. Pediatr. Res.. 11: 453 (1977). celb appeared to have a lower molecular weight than that in 3. Chan, W. Y.. Garnica, A. D., and Rennen 0.M.: Studies of -Cu and lmCd efflux and uptake in Menkes kinky hair (MKHS)fibroblasts. Pediatr. Res., II: normal cells. 453 (1977). To explore further the potential role of metallothionein in the 4. Chan, W. Y., Garnica, A. D.,and Rennen. 0. M.: Defective metallothioneinand LETTER TO THE EDITOR 205

copper accumula~ionin Menkes kinky hair syndrome (MKAS) fibroblasts. mtlallolhiontins in Menkes kinky hair disease. Clin. Chirn. Acta (in press). American hiety of Human Genetics, San Diego, CA, p. 29A, October 19-22, 7. Garnica, A. D., Chan, W. Y., and Rennert, 0.M.: Role of metallothioneins in 1977. copper transpon in patients with Menkes syndrome. Ann. Lab. Clin. Sci. (in 5. Chan. W. Y.. Garnica, A. D., and Rennert, 0.M.: CeH culture studies in Menkes PI")- kinky hair disease. Clin. Chim. Acta (in press). 8. Rennen. 0.M. and Cbn. W. Y.: Menka kinky hair syndrome: is it a reala able 6. Chan. W. Y.. Gamica, A. D..and Rennert, 0. M.: Metal-binding studies of disorder? Clin. Genet., I!: 154 (1977).

Copyrigh~8 1979 lnttrnalional Pediatric Research Foundation, lnc. 003 1 -3998/79/ 1303-0204$02.00/0