Acute Toxicity
Copper toxicity typically results from the production of reactive oxygen species during redox reactions involving excess free or ionic copper forms, and from sufficient accumulation to overwhelm protein-binding capacity (Gaetke et al.
2014). In humans, acute effects of copper ingestion include gastrointestinal symptoms such as nausea or abdominal pain. Olivares et al. (
2001) gave men and women (
n = 61, total) a single dose of 0–12 mg Cu/L as CuSO
4 in deionized drinking water using a randomized design, followed by collection of self-reported perceived symptoms at 15 and 60 min and 24 h. Nausea was most commonly reported immediately following the dose, and 12 mg Cu/L had the highest percentage of nausea reported (21%). The lowest-observed-adverse-effects level (LOAEL) was 4 mg Cu/L, and the no-observed-adverse-effects level (NOAEL) was 2 mg Cu/L. Nausea was reported in 76% of the volunteers.
Studies by Araya et al. (
2001,
2003a) are the most comprehensive investigations of the levels of copper in water that cause acute gastrointestinal effects. These studies were used by ECI (
2008) to derive the LOAEL and NOAEL used for copper in drinking water. Araya et al. (
2001) used a similar study design as reported in Olivares et al. (
2001). Doses of copper were given once a week for 5 weeks at 0–8 mg Cu/L to a total of 179 volunteers (men and women). Up to 18% of volunteers reported nausea at the 8 mg Cu/L dose. In Araya et al. (
2003a), a total of 249 women in Chile, the United States, Northern Ireland, and China were administered a single, oral bolus dose in bottled water at 0, 2, 4, 6, or 8 mg Cu/L. The study was double-blind with each person as their own control. Consistent with the other two studies, nausea was the most common and earliest symptom in Araya et al. (
2003a) and occurred within 15 min of ingestion. Reports of nausea increased with higher doses, and nausea was the most frequently reported symptom. The LOAEL for nausea was 6 mg Cu/L and the NOAEL was 4 mg Cu/L. Other symptoms were reported less frequently and included abdominal pain, vomiting, and/or diarrhea. Gastrointestinal effects occurred immediately and stopped once the exposure ceased (Araya et al.
2003a). These controlled human experimental trials conducted with multiple international populations allow for a reliable identification of a copper concentration in drinking water that does not cause irritation to the gastrointestinal tract leading to symptoms.
Investigation of Liver Toxicosis in Populations Exposed to Elevated Copper
Studies of Indian childhood cirrhosis (ICC) and Tyrolean infantile cirrhosis are the primary reported examples of childhood liver disease resulting from exposure to high copper concentrations along with suspected genetic susceptibility (NRC
2000). In certain regions in India, liver cirrhosis occurred in some infants and young children at a time when milk was stored in copper or brass containers (NRC
2000; Uauy et al.
2008; Nayak and Chitale
2013). High copper concentrations found in the livers of children were quickly reversed with a reduction in exposure and treatment. Clinical features and symptoms of the disease, however, differed from those of Wilson’s disease patients. Moreover, age-matched asymptomatic siblings of ICC cases and control children also showed increases in copper and copper-binding proteins in liver tissue but without the structural and functional changes observed in the ICC cases. With the elimination of storing milk in materials containing copper, the disease frequency decreased, implicating copper in the etiology of the disease, although the association has been noted as circumstantial (Sriramachari and Nayak
2008). It is thought that susceptibility was related to an autosomal recessive trait because some siblings were also affected, whereas the parents of affected children were not affected in childhood (NRC
2000). However, examination of the pedigree of families with ICC and age-matched controls revealed that genetic susceptibility is likely based on multiple factors, rather than a single factor, as no clear evidence of autosomal recessive, partial sex linkage, or double recessive traits was found (Naya and Chitale
2013). No clear documentation exists for the amount of copper infants were exposed to in the Indian childhood liver toxicosis case study; however, one study tried to replicate copper concentrations potentially available through the use of brass vessels for milk storage and found up to 6.21 mg Cu/L in milk after 6 h (O’Neill and Tanner
1989), or 0.93 ± 0.1 mg Cu/kg BW/day for an exposure of a child consuming milk at a rate of 150 mL/kg BW/day.
Further investigation of the origin of the disease by a multicenter study in India indicated that copper may not be the cause in all ICC cases and that excessive liver copper may also be the result of hepatic injury from some other factors (Nayak and Chitale
2013; Sriramachari and Nayak
2008). This collaborative study across six research centers was conducted in the 1980s to clarify the cause of ICC (Nayak and Chitale
2013; Sriramachari and Nayak
2008). Based on a review of 885 children with a liver biopsy, 227 cases of definite ICC were compared with 426 non-ICC control cases for use of “copper yielding utensils.” In two research centers, none of the ICC cases used these utensils, compared with less than 2% of controls. In three research centers, use of these utensils in study subjects was 15–20% with no difference between ICC cases and controls. The remaining research center reported greater utensil use that was not statistically different between ICC cases (55%) and controls (52%). About 10% of definite ICC cases had no possible prior source of excess copper exposure. Asymptomatic siblings with some accumulation of copper in their liver (although not as high as ICC cases) showed no evidence of toxicity and had a decline in copper to normal levels after removal of copper-containing utensils. Hepatic liver concentrations increased in the later stages of ICC rather than in the early stages, suggesting, along with the lack of evidence of higher exogenous copper exposure in most ICC cases, that copper accumulation was a result rather than the cause of the disease. More likely etiologies for ICC were thought to be poor diet quality or postpartum herbal supplements given to new mothers and infants that resulted in adverse effects on the liver rather than toxicity from excess exogenous copper exposure (Nayak and Chitale
2013; Sriramachari and Nayak
2008).
In Tyrolean infantile cirrhosis, elevated copper exposure through heating milk in copper pots or the use of copper utensils was associated with liver cirrhosis and 138 pediatric deaths in an area of western Austria between 1900 and 1980 (NRC
2000; Müller et al.
1996; Uauy et al.
2008). The symptoms were indistinguishable from ICC. Liver cirrhosis appeared to run in families, although not all children within a family were affected. Both affected and unaffected siblings consumed the same milk, which was retrospectively estimated to have a copper concentration of 10.5–63.3 mg Cu/L when prepared and boiled for 20 min in brass and copper pots (Müller et al.
1996). By comparison, reported copper concentrations in breast milk range from ~0.2–1 mg Cu/L and concentrations appear to be highest soon after birth followed by a decline during the first year (ATSDR
2004). The siblings without liver toxicosis were unaffected at doses estimated to be 8–49 times higher than the current 1.3 mg Cu/L EPA action level for drinking water.
Other studies have examined whether populations with higher copper levels in drinking water have resulted in liver toxicosis in infants and children. One study was conducted in three towns in Massachusetts from 1969–1991 with 8.5–8.8 mg Cu/L in drinking water (Scheinberg and Sternlieb
1994). A total of 2788 children under the age of 6 were followed during the course of 23 years to determine the total number of infantile deaths and infantile deaths relating to liver disease. A total of 135 deaths occurred but none were attributed to cirrhosis or any type of liver disease, despite drinking water levels that were higher than the 1.3 mg Cu/L EPA action level by 6.5–6.8 times.
A study in Berlin, Germany, tested water samples from 2944 households with infants (Zietz et al.
2003). A subset of 541 infants consuming tap water with composite water concentrations in their household above 0.8 mg Cu/L (maximum 4.2 mg Cu/L) were selected for medical examination. Nearly all of these 541 infants were examined, none of whom was diagnosed with liver disease. A subset (
N = 183) also received an analysis of serum copper concentration, liver enzyme levels, total bilirubin, and ceruloplasmin, the results of which were not associated with copper exposure. Another case study in Germany indicated that symptoms in 22 children exposed to drinking water containing 0.4–15.5 mg Cu/L were attributed to liver toxicosis, though the water concentrations were not measured until a few months after symptoms were observed (IPCS
1998), and, as for ICC, cases of liver toxicosis are not necessarily associated with copper exposure. Of the 22 children, 13 fatalities were recorded, though fatalities could not be linked to a specific copper concentration in water causing the liver toxicosis. Other cases of idiopathic copper toxicosis have occurred in Mexico (Cabrera-Muñoz et al.
2010) and in Japan (Hayashi et al.
2012).
Brewer (
2000) postulated an underlying basis for genetic susceptibility to excess copper exposure may exist among those who are heterozygous for Wilson’s disease. In particular, Brewer (
2000) noted that a few of these individuals show signs of alterations in copper metabolism that appear to approach those with Wilson’s disease. These individuals are not affected at normal copper exposures but are hypothesized to be more sensitive to excess copper accumulation as exposures increase. Although Wilson’s disease is relatively rare (1 in 50,000 or 1 in 100,000 live births), an estimated 1–2% of the worldwide human population is a heterozygous carrier of this disease (Das and Ray
2006; Gromadzka et al.
2010; Gollan and Gollan
1998). Gromadzka et al. (
2010) examined copper metabolism and function parameters in 68 heterozygous carriers of Wilson’s disease and 31 control individuals. Heterozygote carriers had parameters largely within normal limits with three individuals showing ceruloplasmin levels that were slightly lower than the reference level. As a group, ceruloplasmin levels of the heterozygotes were 75% of the control group, but serum copper levels and urinary copper excretion were not statistically significantly different (Fig. 1 in Gromadzka et al.
2010). Gromadzka et al. (
2010) also noted that internal regulation of copper depends on a number of other genetically determined regulatory factors and that any combination of regulatory factors may compensate in deficiencies associated with Wilson’s disease heterozygosity. No additional research has emerged to further evaluate the hypothesis of environmental sensitivity to copper by heterozygote carriers of Wilson’s disease, nor have additional cases of pediatric liver toxicosis with elevated copper exposure been published, despite the common use of copper in consumer products and the greater prevalence of Wilson’s disease heterozygotes than for Wilson’s disease patients.
Concerns for potential susceptibility to liver toxicosis at elevated copper levels led the NRC to recommend that the 1.3 mg Cu/L EPA action level for copper not be increased (NRC
2000). The scientific literature since does not provide additional evidence to better define a level at which liver toxicosis might occur in susceptible subgroups, although the majority of the evidence involves copper levels in water or milk well in excess of the NOAEL levels defined for acute gastrointestinal effects (i.e., 4 mg Cu/L). Exposure to excess copper is most likely through the use of copper piping for drinking water used in the public water supply and in homes (Uauy et al.
2008).
Reproductive Toxicity
For both reproductive and developmental effects of copper, animal studies indicate that effects to offspring occur at doses that also cause maternal toxicity. Information on potential reproductive toxicity of copper derives primarily from a two-generation rat reproductive toxicity study conducted with CuSO
4 (ECI
2008). The source study is unpublished but was undertaken for a European Regulation on Registration, Evaluation, Authorization, and Restriction of Chemicals submission and is summarized in detail in ECI (
2008). The study was conducted under good laboratory practices and in accordance with standard Organization for Economic Co-operation and Development (OECD) test guidelines. Parental generation (P1) male and female Sprague–Dawley rats (30/sex/group) were fed diets containing 0, 25.4, 127, 254, and 381 mg Cu/kg for premating P1 males and 0, 1.92, 9.6, 19.1, and 29.5 mg CuSO
4/kg BW/day for premating P1 females for at least 70 days before mating and, for females, continuing through gestation and weaning at postpartum day 21. Male and female rats from F1 litters (30/sex/group) were randomly selected to continue with dietary treatment until mating and, for F1 females, through gestation and weaning of F2 pups. Clinical observations, body weight, and food consumption were assessed at least weekly throughout the study. The study evaluated sperm quality and quantity, estrous cyclicity, mating, fertility, and implantation parameters, pup survival and development, sex ratio, sexual development, weight and gross pathology of reproductive and other organs, and histopathology of liver, brain, and reproductive organs. No treatment-related effects on any reproductive or developmental parameters were observed in any dose group for any generation. Thus, the NOAEL for reproductive toxicity was the highest dose group of 381 mg Cu/kg (reported as a copper intake of 23.6−55.7 mg Cu/kg BW/day). The ECI (
2008) summary notes decreased spleen weight for P1 females and F1 and F2 male and female weanlings in the highest dose group. Although not a reproductive effect, ECI (
2008) identifies this as a treatment-related adverse effect with a NOAEL of 254 mg Cu/kg (15.2–26.7 mg Cu/kg BW/day). The ECI (
2008) summary of the unpublished study indicated that apparent changes in spleen weight were small (9–15%) and not statistically significant for F1 weanlings, and potentially transient (spleen weight did not differ from controls in F1 adults). Rat spleen weight can be variable; however, the study authors also showed that F1 and F2 weanling spleen weights in this study were similar to historical controls for the laboratory.
The subchronic exposure study conducted by NTP provides additional information relevant to reproductive toxicity (Hébert
1993). In that study, the highest dietary Cu doses at 68 mg Cu/kg BW/day for rats and 536 mg Cu/kg BW/day for mice did not affect male reproductive organ weights, spermatid or spermatozoal measurements, or estrous cyclicity. Other reproductive endpoints were not measured.
No human studies were identified that adequately evaluate copper exposure and reproductive endpoints. ECI (
2008) cites two case-control studies that reported no association between drinking water copper concentrations and pregnancy outcomes, including spontaneous abortions, stillbirths, neonatal mortality, and congenital abnormalities (Aschengrau et al.
1989,
1993). Copper was one among many chemicals evaluated, and no data were available on individual exposures; samples were collected from the public water system, not individual taps. Thus, these studies have limited value for evaluating copper exposure.
Developmental Toxicity
The developmental toxicity study considered to have the most rigorous design is an unpublished report of a one-generation prenatal developmental toxicity study on rabbits with copper hydroxide (details summarized in ECI
2008), conducted following OECD test method 414. This study administered 0, 6, 9, and 18 mg Cu/kg BW/day by twice daily oral gavage to pregnant female New Zealand white rabbits (22/group) on gestational days 7 through 28. Maternal toxicity occurred in both the 9 and 18 mg Cu/kg BW/day dose groups, including weight loss and reduced food intake early in the treatment period. Although some recovery occurred at the end of the study, maternal body weight gain was 31 and 72% lower than controls in the mid- and high-dose groups, respectively, and food consumption was reduced 17 and 30% in the mid- and high-dose groups, respectively. In addition, three deaths and two abortions occurred in the high-dose group, possibly due to hemorrhages or ulcerative damage to the stomach lining observed in these three animals. The appetite suppression and associated weight loss were considered local effects, similar to acute gastrointestinal effects reported in human studies. No treatment-related developmental effects were observed below the maternally toxic doses. Treatment-related visceral abnormalities or skeletal malformations were not observed at any dose level. The incidence of delayed ossification of the skull and pelvis was increased slightly in the high-dose group, and an increased incidence of supernumerary ribs occurred in the mid- and high-dose groups. The skeletal variation findings should be considered secondary to maternal toxicity. Of note, a high incidence of extra ribs was reported for all groups (64, 67, 80, and 87% incidence at 0, 6, 9 and 18 mg Cu/kg BW/day, respectively), with the higher incidence at the two highest doses likely related to maternal toxicity-induced stress. Likewise, delayed skull and pelvic ossification may be a transient state and likely represent a stress response to general maternal toxicity, as has been reported for other skeletal development delays (e.g., delayed ossification, wavy rigs, bent long bones, and bent scapulae) (Carney and Kimmel
2007; Kimmel et al.
2014). The NOAEL for maternal toxicity in this study was 6 mg Cu/kg BW/day with the decreased food consumption and associated decrease in weight gain likely related to the acute gastrointestinal effects of copper from the gavage administration of large, focused, bolus doses of copper.
Several studies in young animals have examined the potential for possible increased susceptibility to liver toxicosis at early life stages because of developing copper regulatory mechanisms (Araya et al.
2005; Bauerly et al.
2005; Fuentealba et al.
2000). Bauerly et al. (
2005) administered daily doses of 0, 0.01, and 0.025 mg Cu/day in a 10% sucrose solution by oral gavage to suckling Sprague–Dawley rat pups followed by weaning to the same diet received by dams during gestation (supplemented to a 13 mg Cu/kg standard diet concentration). Pups from each group were sacrificed on postnatal days 10 and 20 to assess age- and dose-related differences in organ and whole body copper levels and effects on various regulatory proteins and copper transporters. The authors reported reduced absorption with increasing copper dose at both ages. Increased copper exposure did not affect body weight, serum copper levels, or ceruloplasmin levels, although increased copper exposure did increase the liver copper concentration. Older pups absorbed more copper with increased copper supplementation compared with 10-day-old pups, although older rats showed increased adaptive mechanisms as reflected by increased metallothionein with copper dose. Fuentealba et al. (
2000) fed doses of 1500 mg Cu/kg via the diet to young rats from birth until 16 weeks of age and to adult rats for up to 18 weeks. Young rats accumulated more copper in the liver, showed more severe changes in the liver, and had higher serum enzyme activity when compared with adult rats, indicating that at a dose of 1500 mg Cu/kg, young rats were more susceptible to copper-induced liver injury.
Araya et al. (
2005) fed infant rhesus monkeys formula with 0.6 mg Cu/L supplemented with 6 mg Cu/L (6.6 mg/L total;
N = 5) from ages 0 to 5 months. No evidence of clinical toxicity or histological damage of the liver was noted under light microscopy, despite increases in liver copper. Some ultrastructural changes in liver cells (e.g., irregularly shaped nuclei containing condensed chromatin) under electron microscopy were observed after 1 month but were normal at 5 months. Twice as many apoptotic cells were observed at 5 months compared with the control, although the frequency of these cells was low in all animals. Araya et al. (
2005) postulated that the ultrastructural changes observed might be early cellular damage; however, a major limitation of this study was the availability of only one liver biopsy result from each of the four control animals at 2 months old.
Two studies evaluating the developmental toxicity of copper in rodents provide limited information. Lecyk (
1980) reported decreased litter size, decreased fetal weight, and an increase in skeletal malformations in the fetuses of pregnant C57BL and DBA mice fed diets with ~764 or 1018 mg Cu/kg beginning 1 month before conception and continuing through study termination, although no statistical analysis was conducted. The form of CuSO
4 used was not reported, and values are approximated based on CuSO
4·5H
2O. No effects were reported in mice fed diets containing 127–509 mg Cu/kg. ECI (
2008) estimated a copper LOAEL dose of 123 mg Cu/kg BW/day associated with the diet concentration of 764 mg Cu/kg. The study did not report on maternal parameters, so it was not possible to evaluate the potential relationship between developmental effects and maternal toxicity. Haddad et al. (
1991) administered 0 or 0.158% copper acetate (equivalent to 0.05 mg Cu/L) in drinking water to Wistar rats for 7 weeks before mating and during gestation. Treatment was associated with reduced embryonic and fetal growth (decreased yolk sac diameter, crown-rump length, and somite number) and delayed skeletal ossification in multiple locations. Although maternal growth was unaffected, maternal toxicity included histopathological changes in the liver (hepatocyte degeneration, focal necrosis, and inflammatory changes) and kidney (degenerative changes in proximal convoluted tubules) typically associated with copper deposition. This study included only one dose (82 mg Cu/kg BW/day estimated by WHO
2004) with the developmental effects possibly being secondary to maternal toxicity.
Interactions with zinc may affect whether developmental effects occur in laboratory animal studies (Reinstein et al.
1984). In a factorial design experiment, female Sprague–Dawley rats were given combinations of 1, 10, 100, and 1000 mg Zn/kg diet (equivalent to 1.39, 13.9, 139.0, and 1390.0 mg Zn/kg BW/day)
5 and 0.5, 5, 10, and 100 mg Cu/kg in the diet (equivalent to 0.70, 7.0, 13.9, and 139.0 mg Cu/kg BW/day) from the time of mating to birth of offspring. Fetal malformations were only noted for subjects with zinc-deficient diets (1 and 10 mg Zn/kg diet, or 1.39 and 13.9 mg Zn/kg BW/day), and the rate of malformations increased as the copper level increased when given with the zinc-deficient diet. It was determined that zinc has an antagonistic role in the diet when provided with copper (Reinstein et al.
1984).
Neurological Disorders
Copper is an essential metal that has critical roles in brain development and function. Studies suggest that the dysregulation of copper and related metabolic disorders may also result in neurodegenerative diseases through copper-regulated mechanisms. Cellular respiration and free radical defense mechanisms rely on activities of the copper-requiring enzymes, such as cytochrome C oxidase and Cu, Zn-dependent superoxide dismutase 1 (SOD1). Though not entirely elucidated, copper appears to play a role in amyotrophic lateral sclerosis caused by increased free radical generation possibly linked to a gain of function in SOD1 (Zheng and Monnot
2012). While oxidative damage related to copper and other metal ions is considered an important aspect of neurodegenerative disorders like Alzheimer disease (AD), the role of copper is controversial. The dysregulation of metals such as copper, iron, and zinc has been implicated in oxidative stress and amyloid plaque formation, two common components in AD (Cheignon et al.
2018).
The majority of the knowledge related to copper dysregulation in neurodegenerative diseases stems from AD research for which AD patients display altered regulation of copper and other metals. Key areas of controversy for copper and AD have been whether: (1) excess or deficient copper exposure causes AD and associated pathogenesis, or (2) AD causes copper dysregulation and thereby excess or deficient copper states and neurodegeneration. While age and likely high lipid intake are the greatest risk factors for developing the disease, AD is multifactorial and complex, likely with many genetic and environmental contributing factors, and these issues regarding copper have yet to be resolved (Kardos et al.
2018).
The metal–ion excess hypothesis originated from (1) enriched levels of transition elements Fe, Zn, and Cu in amyloid plaque deposits (mainly consisting of the amyloid β peptide; Aβ) in AD (Atwood et al.
2018; Dong et al.
2003; Lovell et al.
1998) and (2) reports that synthetic Aβ aggregates into fibrils upon binding of copper ions (Bush et al.
1994). Copper’s role in plaque accumulation and neuroinflammation may also be influenced by its accumulation specifically in the brain capillaries (Singh et al.
2013). It has also been reported from in vitro studies with murine macrophage BV2 cells and in a mouse model that copper increases the inflammatory response in the brain, which may affect the impairment of plaque clearance (Kitazawa et al.
2016). In triple transgenic 3xTg-AD mice, chronic copper exposure at 250 mg/L (85 mg/kg BW/day
6) accelerated not only amyloid pathology but also τ pathology in the brain (Kitazawa et al.
2009). Another study has shown an increase in amyloid-β plaques in the brains of Alzheimer’s patients related to a higher generation of radicals when amyloid-β sugars are in the presence of copper (Fica-Contreras et al.
2017).
At the same time, the disturbed bioavailability of copper resulting in deficiency is another feature of AD (Kaden et al.
2011), although the mechanisms and the causal relationships of the reduced copper availability in AD are not well understood (Kessler et al.
2006; Schafer et al.
2007; Klevay
2010; Bost et al.
2016; Bulcke et al.
2017; Li et al.
2017; Bagheri et al.
2018; Kardos et al.
2018). AD-associated alterations in metal–ion (primarily copper) homeostasis were found in all regions of AD brain tissue (Xu et al.
2017), suggesting a pan-cerebral copper deficiency in AD. Such a widespread brain-Cu deficiency may contribute to the pathogenesis by acting through the loss of enzyme functions in energy utilization and antioxidant defenses (Xu et al.
2017). Reduced copper bioavailability to the brain may be further enhanced by the accumulation of copper in AD plaques (Zheng and Monnot
2012), lipid rafts (Bagheri et al.
2018), or astrocytes (Kardos et al.
2018). Research suggesting that copper supplementation may be beneficial in AD include animal models either overexpressing amyloid precursor protein (APP) or APP in combination with other genes like presenilins and τ, or APP in APLP knockout mice. In the latter, copper levels were found to be increased in cerebral cortex and liver (White et al.
1999). Overexpression of APP resulted in significantly reduced brain copper levels in three different transgenic lines (Maynard et al.
2002; Bayer et al.
2003; Phinney et al.
2003). In APP23 mice which overexpress β-APP, supplementation with 65 mg/L Cu(II) in drinking water (administered as copper sulfate pentahydrate) increased brain copper levels, restored superoxide dismutase, lowered β-amyloid peptide levels, and reduced premature deaths (Bayer et al.
2003). Toxic-milk (txJ) mice with a mutant ATPase7b transporter favoring elevated Cu levels when crossed with single transgenic (Tg) CRND8 APP mice, at 6 months of age with 30 mg Cu/kg in the brain showed a reduced number of amyloid plaques and diminished plasma Aβ levels compared with homozygous TgCRND8 mice and to TgCRND8 APP controls (Phinney et al.
2003). Encouraged by the animal studies exhibiting a beneficial outcome of copper treatment, oral supplementation with Cu(II) was investigated in a clinical trial (Kessler et al.
2008). Based on indications of copper deficiency (i.e., lower copper and ceruloplasmin-bound copper levels in plasma of AD patients with advanced biomarkers of the disease in cerebrospinal fluid; Kessler et al.
2006), Kessler et al. (
2008) studied the effect of daily exposure of up to 8 mg Cu(II) over 12 months in AD patients. Plasma copper levels declined in the placebo group, but stabilized in the Cu-treatment group. The treatment did not affect zinc or non-ceruloplasmin levels, although copper had neither a detrimental nor a beneficial effect on AD.
Conversely, several studies have reported the higher levels of total copper and non-ceruloplasmin-bound copper in the serum of AD patients compared with controls (Bagheri et al.
2018). Squitti et al. (
2018) reported the higher levels of non-ceruloplasmin-bound copper and the similar levels of ceruloplasmin-bound copper in the serum of 385 AD patients compared with 336 healthy controls. Serum levels of non-ceruloplasmin-bound copper of AD patients were also similar to those of nine Wilson’s disease patients, although Wilson’s disease patients had lower levels of total copper and ceruloplasmin-bound copper than AD patients. Squitti et al. (
2002) and Brewer et al. (
2010b) attribute the dysregulation of copper in the brains of AD patients to an increase in the “labile” or exchangeable pool of copper in peripheral circulation as represented by non-ceruloplasmin copper. However, the exact chemical nature of this “labile” pool has remained undefined (Ackerman and Chang
2018). Ceruloplasmin is the main copper carrier in plasma (Harris et al.
1999), although many other proteins such as albumin have high bind affinity and capacity for copper and provide reserve binding capacity to keep extracellular ionic copper low. Albumin-bound copper can be delivered to cells and exchanged with other proteins such as transcuprein or with chelators (Linder et al. 2016), and, as noted in the toxicokinetics section, also contributes copper to ceruloplasmin.
As further hypothesized by Brewer (
2010a,
2019), increased divalent copper (Cu(II)) absorption from drinking water or supplements is thought to contribute to this labile pool because divalent copper is not taken up in the gastrointestinal tract by the Ctr1 transporter at the apical membrane of the gastrointestinal tract epithelia cells and processed by the liver, including binding to ceruloplasmin, but instead is rapidly passively absorbed thereby contributing to the non-ceruloplasmin-bound, “labile” copper that can more freely enter the brain. Nevertheless, as noted in the toxicokinetics section, metalloreductases readily reduce Cu(II) for transport by Ctr1 and possibly by a distinct saturable transporter for gastrointestinal uptake of Cu(II) (Lee et al.
2002), and passive absorption appears to be limited even under conditions that would facilitate rapid absorption (e.g., administration of Cu(II) in liquid to participants in a fasting state; Hill et al.
1986; see the toxicokinetics section). Cu(II) also rapidly shifts to Cu(I) in the absence of oxygen (and vice versa).
7
Direct evidence in humans consuming excess copper in drinking water do not support the hypothesis that Cu(II) in water elevates non-ceruloplasmin copper and the more “labile” pool of copper. Adult men and women (48–49 individuals per dose group) drinking <0.01, 2, 4, or 6 mg Cu/day in water for 2 months showed no differences in levels of non-ceruloplasmin copper or parameters reflecting copper loading including red blood cell copper, monocyte copper, superoxide dismutase, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, and serum gama-glutamyltransferase (Araya et al.
2003b). Thus, for the drinking water levels examined, no effect on non-ceruloplasmin-bound copper or copper status was found. Klevay (
2010) has also noted that a higher proportion of non-ceruloplasmin-bound copper in AD patients may occur as a result of decreased ceruloplasmin levels in blood from copper deficiency.
Vascular factors such as hypertension, hypercholesterolemia, and diabetes as well as the inheritance of the epsilon4 allele of the ApoE gene are risk factors for AD (Sjögren and Blennow
2005). Thus, interactions of high fat diet and copper on the risk of AD have been a research interest. An animal model of hypercholesterolemic rabbits showed amyloid-inducing effects at very low levels of copper in drinking water (0.12 mg Cu/L) (Sparks
2004), with similar results reported in other susceptible species/strains (spontaneously hypercholesterolemic rabbits, dogs on a 4% cholesterol diet, transgenic mice with elevated brain amyloid-β; Sparks et al.
2006). Surprisingly, these findings were made only when copper sulfate was added to distilled water, and not when tap water was used in combination with high cholesterol feed (Sparks and Schreurs
2003). Potable tap water typically contains at least an amount of Cu of 0.12 mg Cu/L as copper carbonate or copper carbonate hydroxide (Kaden et al.
2011) which suggests that such effects occur only in the absence of other minerals normally present in tap water. In the rabbit model, extremely high dietary cholesterol levels of 1–2% leads to serious liver toxicity (steatosis and fibrosis; Buyssens et al.
1996; Xu et al.
1997; Schreurs
2013), thereby affecting copper regulation and homeostasis. Thus, the relevance of these findings have serious limitations as a model for human exposure to copper in tap water because of the unrealistically high cholesterol levels and likely involvement of liver impairment affecting copper regulation along with the absence of normal constituents in tap water. Measurements of liver effects, copper uptake, organ distribution, and excretion in comparison with normal diets versus high cholesterol in feed, would enable a better assessment of potential links between hypercholesterolemia and the risk to develop an Alzheimer-like pathology.
A prospective cohort study of 602 older individuals reported that copper supplements in conjunction with a high saturated and trans-fat diet was associated with cognitive decline (Morris et al.
2006). No effect was found for a high cholesterol diet, and AD was not examined. As cautioned by the authors, the multiple comparisons and potential effects of uncontrolled factors (and chance findings), limit causal conclusions. A randomized controlled trial would provide more clarity of the role of copper supplements. No follow up on the role of copper supplements on AD or cognitive decline has been published in the various large prospective cohort aging studies, although these studies have examined many other dietary nutrients (e.g., Harris
2012). In the Iowa Women’s Health Study, Musuru et al. (
2011) reported an increased risk of total mortality in older women who took multivitamins, vitamin B6, folic acid, iron, magnesium, or copper, and a decreased risk for calcium. However, unlike for iron and calcium, associations for copper and risk of mortality were only statistically significant at the first follow up and not at the second and third, and no dose-response for copper and risk of mortality was found.
Similar to the use of copper supplementation to treat AD-related copper deficiency, copper chelation therapy has been proposed for AD based on theories of the effects of a labile pool of excess copper causing AD and the higher levels of serum levels of copper in AD patients in some studies. However, studies that investigated the role of APP and related APLPs in copper homeostasis strictly contradicted a proposed metal chelation based on a better understanding on the molecular level. Compounds like clioquinol, believed to deplete copper levels in the body (Cherny et al.
2001), were shown to drastically increase the intracellular copper concentration in cells (Treiber et al.
2004). Not surprisingly, Drew (
2017) concluded that copper ion chelation therapy has not been effective and should be abandoned. Nevertheless, Squitti et al. (
2017) commented that such therapies have not been fully tested and that a portion of AD patients may benefit. Copper chelator compounds may instead act therapeutically by changing the distribution of copper or facilitating copper uptake (Treiber et al.
2004).
Iron also plays a role in copper regulation in the brain (Monnot et al.
2011). For example, iron deficiency leads to greater copper transport across the blood–brain barrier and promotes copper overload in the central nervous system, though the blood-cerebrospinal fluid barrier is also able to remove excess copper from the cerebrospinal fluid (Monnot et al.
2011). Iron deficiency led to a 70% increase in cellular copper retention and was mediated by multiple transporters and enzymes (Ctr1, DMT1, ATOX1, and ATP7A) (Monnot et al.
2012). Some of the neurological effects could thus be related to interaction and imbalance associated with other essential elements such as iron (Bandmann et al.
2015).
Copper has also been connected to Parkinson’s disease, Huntington’s disease, and autism spectrum disorder (ASD), although these links are controversial and not well understood. Copper may play a role in the pathogenesis, or copper deficiency may be an indicator of those at risk for Parkinson’s (Bulcke et al.
2017). Copper accumulation in brains of Huntington’s disease patients has been implicated in accelerating disease progression; copper chelators and a reduction in copper in the diet was shown to delay the disease progression in animal models (Bulcke et al.
2017). There is some evidence of an association of copper and ASD. Copper levels in plasma of individuals with some forms of ASD were significantly higher than neurotypical individuals and therapy with zinc and B6 reduced copper plasma levels and some ASD symptoms (Russo and deVito
2011); however, the cause(s) of ASD is (or are) currently unknown but thought to be linked to both genetic and environmental factors.
Although copper homeostasis in the brain may be altered at various neurological disease conditions, the available experimental studies in healthy human subjects does not support the view of copper dysregulation due to environmentally relevant exposures to copper. Whether copper exposure affects the risk of AD, under what conditions, and at what levels in susceptible individuals has yet to be established. In general, it appears that copper at environmentally relevant exposures would be well handled by the large majority of the population.