Life Sciences Vol . 19, pp . 1913-1920, 1976 . Printed in the U.S .A.
Pergamon Press
A 3PUDY OF COPPER TRFATMBNT AND TISSUE COPP~t LEVBt+B IN THE MURINS CONGENITAL COPPER DEFICIEAICY, M07.RLED D.M . Hunt School of Biological Sciences, Department of Plant Biology & Microbiology, Queen Mary College, University of London, Mile End Road, London, 81 4NS, England . (Receivnd in final form November 3, 1976) Summary The injection of copper chloride overcomes the lethality and pigment deficiency in the brindled U+1o~ ) mouse mutant but copper levels remain depressed in the liver and brain, and a further accumulation occurs in the kidney . The wpper-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 heterozygotea are reported and in .each case, the changes exceed those expected on the basis of X-inactivation . The aignificance .of these results to the development of a satisfactory treatment regime for this disease is discussed .
An animal model of the human congenital sapper deficiency, Menkes' steely hair disease (1), is provided by the X-linked mottled mutants in the mouse (2) . The accumulation of copper in the intestinal gall and in the kidney and the deficiency elsewhere (2, 3) suggests that copper transport may be defective . Attempts have been made to bypass the gut uptake system in Menkes' infants by the intrnvenoua or infra-muscular administration of copper (4-7) but the results of such treatmeate have been largely disappointing and the administration of a copper-SDTA complex (8) has also proved ineffective . The failure of such treatment ie hoarever not surprising is view of the probable cell-autonomous expression of the disease. Female heterozygotea for the marine disease show a phenotypic variegation in picFnent synthesis (9) and hair structure (10) in the coat following X-inactivation and the clonal inheritance of the inactivated state (11) . The expression of the copper deficiency therefore moat be oellsutonomous in mutant clones . A similar conclusion can be reached for the human disease from the report of pigment variegation in the skin of an older sister of an affected male infant in a negro family (12) . The popper deficiency in mottled mice gives rise to a number of different phenotypic effects . Growth rate is considerably reduced (13), pigment synthesis is restricted to the tip of each hair, and hair structure, especially as evi denced by the presence of curly whiskers, is abnormal (10) . A neurological disturbance of ataxia and tremor is also apparent and this is probably associated with a reduction in a number of copper dependent brain functions, particularly the reduced synthesis of noradrenaline (13, 14) and the reduced activity of cytochrome c oxidase and Superoxide dismutasa (3) . Viability is 1913
1914
Copper Deficiency in Mottled Mice
Vol . 19, No . 12
affected although this is dependent on the particular mottled allele (2) . The work reported in this publication has been largely restricted to the brindled (Mobr ) allele . Brindled males die at 14 days after birth, at an approximately equivalent physiological and developmental age therefore to the age of death in Menkes' infants . The mottled mutants in the mouse provide an excellent system for an asseaament of the effectiveness of copper treatment . The tissue distribution of exogenous copper can be estimated and the effects on survival, pigment synthesis and the activities of copper-dependent enzymes can be determined directly . This publication reports the outcome of copper treatment in mottled mice, together with a study of tissue copper levels in mottled heterozygotes and in males hemizygous for the viable blotchy ( Moblo) allele . Materials and Methods Mutant melee, heterozygous females and normal animals were obtained from matinga between heterozygous females and normal males . Animals were maintained on a normal breeding diet . Copper treatment consisted of 5 uq/q cupric chloride injected intrnperitoneally (i .p .) . Animals were killed by decapitation and tissues stored at - 20°C until required for copper and enzyme analysis . Tissue copper levels ware determined after solubilization in 0 .21 Triton X-100 in water bY flameleas atomic absorption with a Perkin-81mer heated graphite furnace attachment to a Model 303 atomic absorption spectrophotometer . Isotopic copper64 was administered to adult blotchy males and normal 1lttermates in the drinking water at a concentration of 3 .3 yg/ml . Animals were killed after 2 days and the copper 64 content of tissues determined with out further sample preparation in a Nuclear~hicago gamma counter . Cytochrome c oxidase activity and brain noradrenaline content were determined as previously published (2, 3) . Results Aa detailed in Fiq . 1, the i .p . injection of 5 uq/q cupric chloride overcomes the lethality in brindled males but a depression in growth rate, with an actual weight loss between 14 and 23 days, is still evident . The resulting growth curve is closely similar to that found ~or the viable blotchy allele (Fig . 2) . Copper treatment also results in fï darkening of the coat, indicating that the defect in pigment synthesis is at least partially reversed and microscopic examination of individual hairs reveals a resumption in pigment production in the hair shaft . A reversal of the defect in hair structure is indicated by the straightening of the whiskers . The distribution of copper 3 days after a single i .p . injection of 5 uq/q cupric chloride was assessed in brindled males and normal littermatea (Table 1) . Copper treatment fails to eleviate the copper deficiency in mutant liver and brain tissue, although there is a further increase in the already elevated mutant kidney concentration . In contrast, in normal mice the kidney shows no evidence of the copper injection .
Vol . 19, No . 12
Deficiency in Mottled
Copper
Mice
1915
1
f1
11
11
,1
~ Yti~
N
11
N
N
11
1
Fig. 1 Gx»wth curves for copper and water injected hemizygous brindled males (Mobr ) and uninjected normal (+) littermates . Animals were injected i .p . with 5 u4/9 cupric chloride on days 4, 7, 11, 14 and 21 .
1
N
M
N
M. Y ~.
t
N
N
11
Fiq. 2 Growth curves for hemizygous blotchy males (Mobl°) and normal (+) littermates .
TABL$ 1 Hody Distribution of Injected Cupric Chloride in Brindled (Mohr/-) Malaa and Normal (+) Littermates Treatment Kidney
Liver
Brain
Water
Copper
t change in copper content
+ ~br/_
5 .6510.32(5) 17 .78±2 .06(5)
5 .5530 .34(7) 36 .2811.44(5)
-1 .8 +109 .5**
+ Mohr/-
49 .9915.27(4) 8.28±0 .70(4)
112 .86119 .82(7) 12 .34± 1 .14(5)
+125 .8** + 32 .9*
+ Mobr/-
2 .3330.94(4) 1.1530.05(5)
2 .2230 .06(6) 1 .32±0 .08(5)
- 4.7 +14 .8
6 day-old animals were injected with 5 uq/q cupric chloride or water and killed 3 days later. Results are expressed in uq/q ± SSid . *and ** denote aignificance at the 5" and li probability levels respectively . Numbers in parentheses refer to number of animals used . Brain noradrenaline concentrations are Beverly depressed in mutant animals as a wnsequence of a reduction in the _in vivo activity of the copper enzyme, dopamine-ß-hydroxylase (13), and the activity of brain cytochrome c oxidase is The effectiveness of copper treatment in overoominq the also reduced (3) . reduction in the synthetic activity of copper enzymes in brain tissue was followed by examining the ooncentratian of noradrenaLine and the activity of The increase in the level of noradrenaline cytochrome c oxidase (Table 2) .
191 6
Copper Deficiency in Mottled Mice
Vol . 19, No . 12
after wpper treatment indicates that the reduction in dopamine-ß-hydroxylase activity ie entirely reversed . This is not however the case for cytochrome c oxidase activity where the activity in mutant tissue is significantly increased but still falls short of the normal level . The failure of copper treatment to overcome the deficiency in wpper content of liver and brain tissue, together with the observation that the activity of cytochrome c oxidase r~sina depressed, supports the conclusion that the defect in copper homeostasis extends to each and every cell of the body . Further evidence for this interpretation camas from the reduced liver and brain copper contents in heterozygous brindled females (Table 3) . As a result of random X-inactivation female heterozygotes are genetic mosaics containing on average an approximately equal number of normal and mutant cells . The altered tissue copper contents of such mice is compelling evidence for the cell-autonomous nature of the disease . The severity of the changes in copper content were however unexpected, with the liver, brain and kidney copper levels either approaching or equal to those in mutant hemizygotea . TABLE 2 The Effect of Copper Treatment on Brain Noradrenaline Content and Cytochrome c Oxidase Activity in Hrindled (Mohr/ -) Males and Normal (+) Littermates Noradrenaline content Mô r/Water inj .(6)
Copper inj .(6)
1 .3610 .19 probability level
Uninjected (10)
2 .60±0 .41 0 .10
Cytochrome c oxidase activity Mô r/Water inj .(6)
Copper inj .(5)
0 .247±0 .047
0 .31410 .039
probability level
>0 .10
Uninjected (8) 0 .48110 .049
Pergamon Press
A 3PUDY OF COPPER TRFATMBNT AND TISSUE COPP~t LEVBt+B IN THE MURINS CONGENITAL COPPER DEFICIEAICY, M07.RLED D.M . Hunt School of Biological Sciences, Department of Plant Biology & Microbiology, Queen Mary College, University of London, Mile End Road, London, 81 4NS, England . (Receivnd in final form November 3, 1976) Summary The injection of copper chloride overcomes the lethality and pigment deficiency in the brindled U+1o~ ) mouse mutant but copper levels remain depressed in the liver and brain, and a further accumulation occurs in the kidney . The wpper-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 heterozygotea are reported and in .each case, the changes exceed those expected on the basis of X-inactivation . The aignificance .of these results to the development of a satisfactory treatment regime for this disease is discussed .
An animal model of the human congenital sapper deficiency, Menkes' steely hair disease (1), is provided by the X-linked mottled mutants in the mouse (2) . The accumulation of copper in the intestinal gall and in the kidney and the deficiency elsewhere (2, 3) suggests that copper transport may be defective . Attempts have been made to bypass the gut uptake system in Menkes' infants by the intrnvenoua or infra-muscular administration of copper (4-7) but the results of such treatmeate have been largely disappointing and the administration of a copper-SDTA complex (8) has also proved ineffective . The failure of such treatment ie hoarever not surprising is view of the probable cell-autonomous expression of the disease. Female heterozygotea for the marine disease show a phenotypic variegation in picFnent synthesis (9) and hair structure (10) in the coat following X-inactivation and the clonal inheritance of the inactivated state (11) . The expression of the copper deficiency therefore moat be oellsutonomous in mutant clones . A similar conclusion can be reached for the human disease from the report of pigment variegation in the skin of an older sister of an affected male infant in a negro family (12) . The popper deficiency in mottled mice gives rise to a number of different phenotypic effects . Growth rate is considerably reduced (13), pigment synthesis is restricted to the tip of each hair, and hair structure, especially as evi denced by the presence of curly whiskers, is abnormal (10) . A neurological disturbance of ataxia and tremor is also apparent and this is probably associated with a reduction in a number of copper dependent brain functions, particularly the reduced synthesis of noradrenaline (13, 14) and the reduced activity of cytochrome c oxidase and Superoxide dismutasa (3) . Viability is 1913
1914
Copper Deficiency in Mottled Mice
Vol . 19, No . 12
affected although this is dependent on the particular mottled allele (2) . The work reported in this publication has been largely restricted to the brindled (Mobr ) allele . Brindled males die at 14 days after birth, at an approximately equivalent physiological and developmental age therefore to the age of death in Menkes' infants . The mottled mutants in the mouse provide an excellent system for an asseaament of the effectiveness of copper treatment . The tissue distribution of exogenous copper can be estimated and the effects on survival, pigment synthesis and the activities of copper-dependent enzymes can be determined directly . This publication reports the outcome of copper treatment in mottled mice, together with a study of tissue copper levels in mottled heterozygotes and in males hemizygous for the viable blotchy ( Moblo) allele . Materials and Methods Mutant melee, heterozygous females and normal animals were obtained from matinga between heterozygous females and normal males . Animals were maintained on a normal breeding diet . Copper treatment consisted of 5 uq/q cupric chloride injected intrnperitoneally (i .p .) . Animals were killed by decapitation and tissues stored at - 20°C until required for copper and enzyme analysis . Tissue copper levels ware determined after solubilization in 0 .21 Triton X-100 in water bY flameleas atomic absorption with a Perkin-81mer heated graphite furnace attachment to a Model 303 atomic absorption spectrophotometer . Isotopic copper64 was administered to adult blotchy males and normal 1lttermates in the drinking water at a concentration of 3 .3 yg/ml . Animals were killed after 2 days and the copper 64 content of tissues determined with out further sample preparation in a Nuclear~hicago gamma counter . Cytochrome c oxidase activity and brain noradrenaline content were determined as previously published (2, 3) . Results Aa detailed in Fiq . 1, the i .p . injection of 5 uq/q cupric chloride overcomes the lethality in brindled males but a depression in growth rate, with an actual weight loss between 14 and 23 days, is still evident . The resulting growth curve is closely similar to that found ~or the viable blotchy allele (Fig . 2) . Copper treatment also results in fï darkening of the coat, indicating that the defect in pigment synthesis is at least partially reversed and microscopic examination of individual hairs reveals a resumption in pigment production in the hair shaft . A reversal of the defect in hair structure is indicated by the straightening of the whiskers . The distribution of copper 3 days after a single i .p . injection of 5 uq/q cupric chloride was assessed in brindled males and normal littermatea (Table 1) . Copper treatment fails to eleviate the copper deficiency in mutant liver and brain tissue, although there is a further increase in the already elevated mutant kidney concentration . In contrast, in normal mice the kidney shows no evidence of the copper injection .
Vol . 19, No . 12
Deficiency in Mottled
Copper
Mice
1915
1
f1
11
11
,1
~ Yti~
N
11
N
N
11
1
Fig. 1 Gx»wth curves for copper and water injected hemizygous brindled males (Mobr ) and uninjected normal (+) littermates . Animals were injected i .p . with 5 u4/9 cupric chloride on days 4, 7, 11, 14 and 21 .
1
N
M
N
M. Y ~.
t
N
N
11
Fiq. 2 Growth curves for hemizygous blotchy males (Mobl°) and normal (+) littermates .
TABL$ 1 Hody Distribution of Injected Cupric Chloride in Brindled (Mohr/-) Malaa and Normal (+) Littermates Treatment Kidney
Liver
Brain
Water
Copper
t change in copper content
+ ~br/_
5 .6510.32(5) 17 .78±2 .06(5)
5 .5530 .34(7) 36 .2811.44(5)
-1 .8 +109 .5**
+ Mohr/-
49 .9915.27(4) 8.28±0 .70(4)
112 .86119 .82(7) 12 .34± 1 .14(5)
+125 .8** + 32 .9*
+ Mobr/-
2 .3330.94(4) 1.1530.05(5)
2 .2230 .06(6) 1 .32±0 .08(5)
- 4.7 +14 .8
6 day-old animals were injected with 5 uq/q cupric chloride or water and killed 3 days later. Results are expressed in uq/q ± SSid . *and ** denote aignificance at the 5" and li probability levels respectively . Numbers in parentheses refer to number of animals used . Brain noradrenaline concentrations are Beverly depressed in mutant animals as a wnsequence of a reduction in the _in vivo activity of the copper enzyme, dopamine-ß-hydroxylase (13), and the activity of brain cytochrome c oxidase is The effectiveness of copper treatment in overoominq the also reduced (3) . reduction in the synthetic activity of copper enzymes in brain tissue was followed by examining the ooncentratian of noradrenaLine and the activity of The increase in the level of noradrenaline cytochrome c oxidase (Table 2) .
191 6
Copper Deficiency in Mottled Mice
Vol . 19, No . 12
after wpper treatment indicates that the reduction in dopamine-ß-hydroxylase activity ie entirely reversed . This is not however the case for cytochrome c oxidase activity where the activity in mutant tissue is significantly increased but still falls short of the normal level . The failure of copper treatment to overcome the deficiency in wpper content of liver and brain tissue, together with the observation that the activity of cytochrome c oxidase r~sina depressed, supports the conclusion that the defect in copper homeostasis extends to each and every cell of the body . Further evidence for this interpretation camas from the reduced liver and brain copper contents in heterozygous brindled females (Table 3) . As a result of random X-inactivation female heterozygotes are genetic mosaics containing on average an approximately equal number of normal and mutant cells . The altered tissue copper contents of such mice is compelling evidence for the cell-autonomous nature of the disease . The severity of the changes in copper content were however unexpected, with the liver, brain and kidney copper levels either approaching or equal to those in mutant hemizygotea . TABLE 2 The Effect of Copper Treatment on Brain Noradrenaline Content and Cytochrome c Oxidase Activity in Hrindled (Mohr/ -) Males and Normal (+) Littermates Noradrenaline content Mô r/Water inj .(6)
Copper inj .(6)
1 .3610 .19 probability level
Uninjected (10)
2 .60±0 .41 0 .10
Cytochrome c oxidase activity Mô r/Water inj .(6)
Copper inj .(5)
0 .247±0 .047
0 .31410 .039
probability level
>0 .10
Uninjected (8) 0 .48110 .049
