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Blackfoot Disease and Arsenic: A Never-Ending Story


a b
Chin-Hsiao Tseng
a
Division of Endocrinology and Metabolism, Department of Internal Medicine, National
Taiwan University Hospital; National Taiwan University College of Medicine; School of Public
Health, Taipei Medical University, taipei, Taiwan
b
Division of Endocrinology and Metabolism, Department of Internal Medicine, National
Health Research Institutes, taipei, Taiwan

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To cite this article: Chin-Hsiao Tseng (2005): Blackfoot Disease and Arsenic: A Never-Ending Story, Journal of Environmental
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Blackfoot Disease and Arsenic:


A Never-Ending Story
Downloaded by [University of Illinois Chicago] at 23:14 14 July 2012

Chin-Hsiao Tseng
Division of Endocrinology and Metabolism, Department of Internal Medicine, National
Taiwan University Hospital; National Taiwan University College of Medicine; School of
Public Health, Taipei Medical University, Taipei, Taiwan; and Division of Environmental
Health and Occupational Medicine of the National Health Research Institutes,
Taipei, Taiwan
Blackfoot disease (BFD) is an endemic peripheral vascular disease confined to the
southwestern coast of Taiwan. This article reviews the epidemiology, clinical manifes-
tations and diagnosis, pathology, etiology and pathogenesis of this disease. Sporadic
cases of BFD occurred as early as in the early 20th century, and peak incidence was
noted between 1956 and 1960, with prevalence rates ranging from 6.51 to 18.85 per
1,000 population in different villages. Typical clinical symptoms and signs of progres-
sive arterial occlusion mainly found in the lower extremities, but in rare cases, the
upper extremities might also be involved. Ulceration, gangrene and spontaneous or
surgical amputation were typical fate. An extensive pathological study concluded that
30% of the BFD patients had histologic lesions compatible with thromboangiitis oblit-
erans and 70% showed changes of arteriosclerosis obliterans. Epidemiologic studies
carried out since mid-20th century revealed that BFD was associated with the con-
sumption of inorganic arsenic from the artesian wells. Recent studies confirmed the
existence of preclinical peripheral vascular disease, subclinical arterial insufficiency
and defects in cutaneous microcirculation in the residents of the endemic villages.
A more recent study suggested that the methylation capacity of arsenic can inter-
act with arsenic exposure in the development of peripheral vascular disease among
residents of BFD-endemic areas. The incidence of BFD decreased dramatically after
the implementation of tap water in these villages over the past 2–3 decades. The
atherogenicity of arsenic could be associated with its effects of hypercoagulability, en-
dothelial injury, smooth muscle cell proliferation, somatic mutation, oxidative stress,
and apoptosis. However, its interaction with some trace elements and its association
with hypertension and diabetes mellitus could also explain part of its higher risk of
developing atherosclerosis. Although humic substances have also been suggested as
a possible cause of BFD, epidemiologic studies are required to confirm its etiologic
role.

Address correspondence to Chin-Hsiao Tseng, MD, PhD, Department of Internal


Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei,
Taiwan; E-mail: ccktsh@ms6.hinet.net

55
56 C.-H. Tseng

Key Words: Atherogenicity; Blackfoot disease; Inorganic arsenic; Peripheral vascular


disease; Water pollutant.

INTRODUCTION
In a confined area located at the southwestern coast of Taiwan, the prevalence
of a unique peripheral vascular disease (PVD) was high (1, 2). The disease has
been known as blackfoot disease (BFD) after its characteristic progression from
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numbness or coldness of one or more extremities, to intermittent claudication,


and finally to gangrene and spontaneous amputation (1, 2). Extensive epidemio-
logic (1) and pathological (3) studies and water analyses (4, 5) have been carried
out since the 1950s and the etiology of BFD has been linked to the drinking of
artesian well water. Although arsenic has been suggested as the most probable
cause of the disease, some investigators favored other potential etiologic factors
such as humic substances (6). Despite the controversies over its real etiology,
BFD has declined dramatically 2–3 decades after the implementation of tap
water supply and termination of the use of groundwater for drinking in the
endemic areas (2). In this article, the author reviews the disease on the aspects
of epidemiology, clinical manifestations and diagnosis, pathology, etiology and
pathogenesis.

EPIDEMIOLOGY
Since the early 20th century, sporadic cases of a strange disease involving the
lower extremities have occurred, which was characterized by progressive black-
ish discoloration of the skin extending from the toes gradually upward towards
the ankles (1, 2). The patients also suffered from numbness or coldness of the
extremities and intermittent claudication before the development of gangrene.
Ulceration might also be found in the patients. The lesions might progress to
spontaneous amputation in some cases or surgical amputation was required
for saving lives. In rare cases, the fingers might also be involved (3). The mean
age of onset of BFD in a series of 1,300 cases was 52 years, with a sexual ratio
of men to women of 1.5 (1). However, the disease could afflict patients within a
wide range of age, from 2 to 87 years (1).
According to epidemiologic studies, the peak incidence of BFD was found be-
tween 1956 and 1960 (2). Most of the patients resided in a confined area located
along the southwestern coast of Taiwan. More than 72% of the cases occurred
in Hsueh-Chia Township and Pei-Men Village of Tai-Nan County, and Pu-Tai
Township and Yii-Chu Village of Chia-Yi County. These areas were called the
“old endemic areas.” Some cases were also found in the so-called “new endemic
areas” located on the periphery of the “old endemic areas.” These “new endemic
Blackfoot Disease and Arsenic 57
areas” were confined to the counties of Yun-Ling, Chia-Yii, Tai-Nan, and Ping-
Tung. A total of more than 1,600 cases had been discovered up to 1980s. In
a survey carried out from 1984 to 1985, a total of 1,220 prevalent cases (690
men and 530 women) were found in the “old endemic areas.” Among them,
374 (30.7%), 352 (28.9%), 309 (25.3%), and 185 (15.2%) cases were found in
Hsueh-Chia, Pu-Tai, Pei-Men, and Yii-Chu, respectively. The prevalence rates
in Pei-Men, Hsueh-Chia, Pu-Tai and Yii-Chu were 18.85, 11.57, 8.80 and 6.51
per 1,000 population, respectively.
The prognosis of BFD is relatively poor after the onset of clinical symptoms.
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In a series of 1,300 cases, 68% of the patients underwent spontaneous or surgical


amputation (1). Failure of the amputation wound to heal was common and ream-
putation rate was as high as 23.3% (1). The annual death rate of the cases was
4.84 per 100 patient-years and the mean age at death was about 62 years (1).

CLINICAL MANIFESTATIONS AND DIAGNOSIS


Earlier studies carried out in BFD areas in the 1960s to 1980s focused on the
clinically full-blown disease. The diagnosis of BFD was based on clinical findings
of gangrene and/or the presence of symptoms or signs on physical examination,
such as numbness or coldness in the extremities, intermittent claudication, or
absence of peripheral pulsation. According to the study of Tseng (1), the initial
clinical symptoms and signs among 1,070 BFD patients are listed in Table 1.
However, about 13.9% of the patients would have sudden onset of BFD without
symptoms (1). The pathological changes of BFD are basically atherosclerosis
and grossly undifferentiated from atherosclerosis caused by other etiology (3).
Therefore, the diagnosis of BFD was always based on clinical exclusion of other
common causes of PVD.
The basis of the diagnostic criteria used by earlier investigators was more
or less subjective and tended to vary in different environments of examination
and from observer to observer. Because atherosclerosis is an insidious process

Table 1: Initial symptoms/signs among 1,070 patients with blackfoot disease


(adapted from (1)).

Initial symptoms/signs Percentage (%)


Numbness 75.1
Coldness in the extremities 57.0
Intermttent claudication 42.2
Ruborous foot 15.3
Burning sensation in soles 15.0
Pale foot 14.9
Weakness of extremities 10.2
Itching sensation in soles 7.0
58 C.-H. Tseng

and the induction period of BFD is about 20–30 years (2), the circulation of
the lower extremity can be better assessed by more sophisticated laboratory
examinations than its severe clinical manifestations as BFD. Later studies car-
ried out since the 1990s have focused on the use of more objective diagnostic
methods such as the Doppler ultrasound (7, 8), laser Doppler flowmetry (9) and
a combination with exercise test (10). These later studies not only confirmed
a higher rate of PVD in residents of the BFD areas with or without clinical
manifestations of full-blown disease, subclinical arterial insufficiency and mi-
crocirculatory defects were also demonstrated in seemingly normal subjects
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living in the hyperendemic areas.


In order to evaluate the presence of subclinical arterial insufficiency in the
seemingly normal subjects living in these BFD-hyperendemic villages, Tseng
et al. measured the ankle pressure before and after a standardized treadmill
exercise test in 23 men living in the BFD area and 28 men living in a nearby
non-endemic township (10). It has been shown that the seemingly normal sub-
jects living in BFD-hyperendemic villages had an abnormal drop of ankle pres-
sure after exercise, while the control subjects had increased ankle pressure
in correspondence to increased brachial pressure. As a consequence, the dif-
ference between ankle and brachial pressures, or the ankle-brachial gradient,
decreased after exercise in the seemingly normal subjects living in the BFD-
hyperendemic villages, while it remained unchanged in the control group. In
another study that evaluated the cutaneous perfusion of the big toe by laser
Doppler flowmetry, the seemingly normal subjects living in a BFD area had sig-
nificantly lower flow than the control subjects either at 36◦ or after local skin
heating to 42◦ (9). This phenomenon still existed even after excluding those
who probably had minor or subclinical arterial insufficiency. These results in-
dicate the development of early subclinical deficits in arterial circulation and
cutaneous microcirculation before the onset of BFD.

PATHOLOGY
The only pathological study in the BFD patients was done by Yeh and How dur-
ing the late 1950s and early 1960s at the National Taiwan University Hospital
(3). The study reported the pathological findings of a total of 63 limbs taken from
52 BFD patients (35 men and 17 women). The age ranged from 12 to 76 years.
Except for one ring finger taken from a female patient, all others were taken
from the lower extremities. This extensive pathological study concluded that
30% of the BFD patients had histologic lesions compatible with thromboangiitis
obliterans and 70% showed changes of arteriosclerosis obliterans. The common
histological features in the thromboangiitis obliterans group include 1) fibri-
noid necrosis of the whole vessel wall of arterioles, venules or precapillaries;
Blackfoot Disease and Arsenic 59
2) proliferation and activation of vascular endothelium; and 3) diffuse infiltra-
tion of inflammatory cells throughout the whole vessel wall, giving rise to a
picture of nodular non-suppurative, non-thrombotic panarteritis. Histological
features of vessels found in arteriosclerosis obliterans group include 1) throm-
bus formation with newly formed vessels having reduced number and size,
leaving a poorly vascularized, hyalinized dense fibrous tissue like an old scar
tissue; 2) intimal sclerosis; 3) medial calcific change; and 4) adventitial change
of periarterial fibrosis.
Autopsy was performed in 3 of the BFD patients, including two men aged
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39 and 62 years, respectively, and one woman aged 24 years. It was interesting
to note that all of them had generalized atherosclerosis, especially involving
the coronary arteries, even in the young woman. This finding suggested that
the clinical manifestations of BFD were only part of a systemic involvement of
atherosclerosis in the patients.

ETIOLOGY
The etiology of BFD was unknown initially, but after a series of investigations
in the mid-20th century, the suspected cause had been focused on the artesian
well water consumed by the residents of the endemic area. Because of the high
content of salt in shallow well water, residents in these endemic villages have
used water from artesian wells as deep as 100–300 meters since the 1920s (5).
Arsenic, ergot alkaloid, organic chlorides, and humic acid have been identified
in high concentrations from the water (6). In addition, trace elements, such as
molybdenum, mercury, copper, and cadmium, were reported to be detectable in
the serum and/or urine of BFD patients and in groundwater they used (11, 12).
However, extensive epidemiologic studies favor arsenic as the most probable
cause (1, 7, 8). The median arsenic concentration of the artesian well water in
these BFD-hyperendemic villages ranged from 0.70 to 0.93 mg/L (4, 5), while
the shallow well water in other areas had an arsenic content between non-
detectable and 0.30 mg/L with a median of 0.04 mg/L (5). The standard for
arsenic in drinking water set by the World Health Organization is 0.01 mg/L
and 0.05 mg/L by the United States Public Health Service (13). A survey of the
arsenic contents of 83,563 wells all over Taiwan showed that 29.1% of the wells
in BFD-endemic areas had an arsenic content greater than 0.05 mg/L and 5.2%
greater than 0.35 mg/L with the highest value being 2.5 mg/L. In other areas of
Taiwan, only 5.7% of the wells had an arsenic content greater than 0.05 mg/L
and 0.3% greater than 0.35 mg/L (14).
Besides arsenic intake from well water, residents of the BFD areas could
be exposed to arsenic from a variety of other sources. Most residents in the
BFD areas were engaged in farming, fishery and salt production. The arsenic
60 C.-H. Tseng

content of the soil was high and ranged from 5.3 to 11.2 mg/kg with a median of
7.2 mg/kg (14). While artesian well water was extensively used for agricultural
and piscicultural purposes, the residents could also be exposed to arsenic from
farm products and fish (15). The amount of arsenic ingested by residents in
BFD-hyperendemic areas was estimated to be as high as 1 mg per day (16).
The lethal dose in human beings is estimated to be 1 mg/kg/day (17).
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LINK BETWEEN ARSENIC EXPOSURE AND BFD


Because of the finding of high arsenic content in the artesian well water, Tseng
(1) evaluated the association between the arsenic concentration in well water
and the prevalence of BFD in the endemic villages. A dose-responsive relation-
ship between them was demonstrated in different age groups of residents. In
villages where the arsenic concentrations in well water were <0.30 mg/L, 0.30–
0.59 mg/L, and more than 0.60 mg/L, the prevalence rates of BFD for residents
aged 20–39 years were 0.5%, 1.3%, and 1.4%, respectively; for residents aged
40–59 years, 1.1%, 3.2%, and 4.7%, respectively; and for residents aged over
60 years, 2.0%, 3.2%, and 6.1%, respectively (1). This study suggested that the
prevalence of BFD increased with regards to increasing arsenic concentrations
of the well water, which could not be explained by the difference of age.
Some criticisms for this early study arose with regards to the facts that
the diagnosis of BFD was more or less subjective, arsenic exposure index did
not take the duration of exposure into account and did not consider exposure
dosage at individual levels, and the important risk factors for PVD such as
dyslipidemia, smoking, hypertension, diabetes mellitus, etc. were not adjusted
for in the evaluation of a cause-effect relationship. To further clarify the effect of
arsenic exposure on the development of PVD among the residents of the BFD
areas, Tseng and colleagues carried out a series of studies by using Doppler
ultrasound as a diagnostic tool, calculating indices for estimating individual
dosage of arsenic exposure, and controlling possible confounders for PVD (7, 8).
Dose-responsive patterns between indices of long-term arsenic exposure and
PVD could be clearly demonstrated. The prevalence rates of PVD for those with
a cumulative arsenic exposure (CAE) of 0, 0.1–19.9 and ≥20 mg/L-years were
4.4, 11.6 and 19.8%, respectively; the respective odds ratios were 1.00, 2.77
(0.84–9.14) and 4.28 (1.26–14.54) after adjustment for potential confounders
(2). The prevalence of PVD for those living in the endemic areas for ≥60 years
could be as high as 28.4% and the multivariate-adjusted odds ratio was 10.54
(2.68–41.37) while compared to those living in the endemic areas for less than
40 years (2). These later studies fortified the link between arsenic exposure and
development of PVD in the BFD-endemic areas in Taiwan.
Because only a small proportion of the exposed residents would develop
PVD, individual capacity to metabolize and detoxify the ingested inorganic
Blackfoot Disease and Arsenic 61
arsenic was believed to play an important role on disease development. This
individual susceptibility may involve genetic and/or acquired factors. Recently,
Tseng and colleagues examined the interaction between arsenic exposure and
urinary arsenic species on the risk of PVD diagnosed by Doppler ultrasound
in 479 (220 men and 259 women) adults residing in the BFD areas in Taiwan
(18). Arsenic exposure was estimated by CAE and urinary levels of total ar-
senic, inorganic arsenite and arsenate, monomethylarsonic acid (MMAV ) and
dimethylarsinic acid (DMAV ) were determined, and primary methylation index
(PMI = MMAV /urinary inorganic arsenic) and secondary methylation index
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(SMI = DMAV /MMAV ) were calculated. The association between PVD and uri-
nary arsenic parameters was evaluated with consideration of the interaction
with CAE and the confounding effects of age, sex, body mass index, total choles-
terol, triglycerides, cigarette smoking and alcohol consumption. Results showed
that PVD risk increased with a higher CAE and a lower capacity to methylate
arsenic to DMAV after taking into account the potential effect of confounders.
The multivariate-adjusted odds ratios for CAE of 0, 0.1–15.4 and >15.4 mg/L ×
year were 1.00, 3.41 (0.74–15.78) and 4.62 (0.96–22.21), respectively ( p < 0.05,
trend test); and for PMI ≤ 1.77 and SMI > 6.93, PMI > 1.77 and SMI > 6.93,
PMI > 1.77 and SMI ≤ 6.93, and PMI ≤ 1.77 and SMI ≤ 6.93 were 1.00, 2.93
(0.90–9.52), 2.85 (1.05–7.73) and 3.60 (1.12–11.56), respectively ( p < 0.05, trend
test). It was concluded that individuals with a higher arsenic exposure and a
lower capacity to methylate inorganic arsenic to DMAV have a higher risk of
developing PVD in BFD areas in Taiwan. This is the first study showing an
effect of arsenic methylation capacity on the development of arsenic-induced
PVD. Therefore, the results of recent studies suggested that PVD susceptibil-
ity is not only related to the exposure dosage of arsenic in the BFD areas, the
metabolism of arsenic has a significant and great impact on the susceptibility
and development of PVD in subjects chronically exposed to arsenic: the more
efficient to methylate to DMAV , the lower the risk.
The association between arsenic exposure and BFD is also supported by the
observation that patients with BFD had a high co-occurrence of arsenic-related
skin lesions such as hyperpigmentation, hyperkeratosis, and skin cancer (19)
and a higher risk of sensory neuropathy (20), which is well known for acute
arsenic intoxication. The co-occurrence of skin lesions and higher risk of neu-
ropathy among the residents of BFD areas could not be attributed to chance
alone, and chronic arsenic exposure could play an important etiologic role.
The different rates of BFD between residents consuming well water and
those consuming tap water in the same areas and the dramatic decline of BFD
after implementation of tap water supply system to the endemic villages also
support arsenic from the artesian well water as a cause of BFD. Tap water
supply to the endemic areas was not available before the 1960s and its coverage
remained low until the 1970s. The incidence rates per 100,000 person-years
for men and women were 44.3 and 36.5, respectively, for those who consumed
62 C.-H. Tseng

artesian well water; and were 2.9 and 3.1 for men and women, respectively, for
those who used tap water in the same areas (2). Most new cases of BFD after
the 1970s occurred in people above 50 years of age in both genders, while BFD
might occur in those below 30 years of age before the 1950s (2). The occurrence
of BFD decreased dramatically over the past 2–3 decades after tap water supply
was implemented in the endemic region (2). A recent study also showed that
ischemic heart disease mortality declines gradually about 17 to 21 years after
the cessation of consumption of high-arsenic artesian well water in the BFD
areas, which is contrary to an increasing trend of cardiovascular mortality in
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the general population of Taiwan (21). These findings strengthen the likelihood
that an association exists between arsenic exposure and the development of
atherosclerotic diseases.
Another evidence supporting the arsenic hypothesis was the similar obser-
vation of arsenic-induced PVD among residents in Chile and Mexico, Moselle
vintners, and copper smelter workers (22). Residents in the BFD areas also have
a significantly increased risk of (23, 24) and mortality from (24, 25) ischemic
heart disease and stroke (26), which were also correlated with arsenic exposure
in a dose-responsive pattern. These findings were compatible with the patholog-
ical findings of severe generalized atherosclerosis in BFD patients as shown by
Yeh and How (3), and with the finding of cardiovascular disease representing a
major cause of death (44%) in the BFD patients (1). Therefore, arsenic-induced
atherosclerosis can be consistently demonstrated in epidemiologic studies and
the process is systemic.
A recent study treating ApoE−/− /LDLr−/− mice with 133 µM (10 ppm)
sodium arsenite in drinking water for 18 weeks has successfully induced a
significant increase in atherosclerotic plaques in the innominate artery while
compared to controls (27). This animal model provided evidence for biological
plausibility of arsenic-induced atherosclerosis observed in humans in epidemi-
ologic studies.

PATHOGENETIC MECHANISMS OF ARSENIC-INDUCED


ATHEROSCLEROSIS
The mechanisms of arsenic-induced atherosclerosis still remain to be eluci-
dated. The potential mechanisms have been reviewed recently (2, 28). Some of
the characteristics of arsenic can explain its effect of atherogenicity (Table 2).
Arsenic is genotoxic (29) and has been demonstrated to cause damage to en-
dothelial cells (30), to cause endothelial dysfunction (31), to increase coagu-
lability (32), to decrease fibrinolysis (33), to induce oxidative stress with im-
paired nitric oxide balance (34, 35), to enhance inflammatory activity (36), to
promote apoptosis (37), and to stimulate smooth muscle cell proliferation (38).
Arsenic can also interact with other trace elements (39–44) and has been found
Blackfoot Disease and Arsenic 63
Table 2: Pathogenetic mechanisms of atherogenicity induced by arsenic.

Endothelial cell damage


Endothelial cell dysfunction
Increased inflammatory activity
Increased coagulability and decreased fibrinolysis
Smooth muscle cell proliferation
Interference with functions of DNA, RNA, and proteins
Increased oxidative stress with impaired nitric oxide balance
Induction of apoptosis
Interaction with other trace elements
Association with hypertension and diabetes mellitus
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in epidemiologic studies to be associated with hypertension (45) and diabetes


mellitus (46–49). All of these could trigger, predispose, or aggravate the process
of atherosclerosis.
Arsenic has been shown to induce endothelial dysfunction (31), peroxyni-
trite generation and cyclooxygenase-2 protein expression in endothelial cells
(50, 51). Arsenic also induces expression of genes coding for inflammatory
mediators including IL-8 in human aortic endothelial cell (36). Some recent
studies demonstrated that arsenic induces antioxidative enzymes, including
heme oxygenase-1, thioredoxin peroxidase-2, NADPH dehydrogenase, and glu-
tathione S-transferase P subunit, suggesting the induction of oxidative stress
by arsenic (35). Our previous study demonstrated the existence of microcir-
culatory defects in seemingly normal subjects living in the BFD-hyperendemic
areas (9). A recent in vitro study confirmed that exposure of human microvascu-
lar endothelial cells to arsenic resulted in a decrease of tissue-type plasminogen
activator and an increase in plasminogen activator inhibitor type-1 expression
as well as reduced fibrinolysis, which were not likely shown in the macrovas-
cular endothelial cells (33). An endemic area of chronic arsenic poisoning and
experimental animal studies elucidated a potential in vivo impairment of nitric
oxide formation and oxidative stress caused by prolonged exposure to arsenate
in the drinking water (52) Bunderson and colleagues (27) reported that changes
in specific inflammatory mediators such as leukotriene and prostacyclin are re-
lated to arsenic-induced atherosclerosis.
Recently, the key metabolic intermediates of inorganic acid, monomethylar-
sonous acid (MMAIII ) and dimethylarsonic acid (DMAIII ), have been identified
in human urine (53–55). Many studies also demonstrated that the triva-
lent methylated arsenic species are more toxic than the pre-methylated inor-
ganic compounds. MMAIII and DMAIII could exhibit properties of inhibition on
cysteine-containing enzymes (56), cellular toxicity (57), genotoxicity, and clas-
togenicity (58). In addition, MMAIII is a potent and irreversible inhibitor of an
enzyme involved in cellular response to oxidative stress (59). The DNA damage
induced by methylated trivalent arsenicals can either be direct (60) or medi-
ated by the reactive oxygen species formed concomitantly with the oxidation
64 C.-H. Tseng

of DMAIII to DMAV (61). DMAIII can produce dimethylarsinic peroxyl radical


and dimethylarsinic radical (62). Arsenic is diabetogenic (46–49) and a recent
study has clearly demonstrated that insulin-dependent glucose uptake by 3T3-
L1 adipocytes is also significantly inhibited by trivalent arsenicals, either in
inorganic form or in methylated form (63).
Cancers can be caused by a variety of risk factors and environmental ex-
posure (64–70). A dual effect of carcinogenicity and atherogenicity has been
noted from exposures to a variety of substances, such as arsenic, ionizing ra-
diation, vinyl chloride monomer, tobacco, and industrial combustion effluents
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containing polycyclic aromatic hydrocarbons (71). Somatic mutations are as-


sumed to be the basic pathogenesis for the co-occurrence. Benditt and Benditt
(72) have observed monotypic cells in atherosclerotic plaques in heterozygotes
of glucose-6-phosphatase. Monoclonal origin has also been found to be a char-
acteristic of most cancers. This identical feature of monoclonal origin implies a
possible common etiology involving somatic mutation and cell proliferation in
atherosclerosis and cancer (71). Several genes may be involved in the multifac-
torial pathogenesis of atherosclerosis. Increased mutation rate has also been
shown to be involved in the formation of atherosclerotic plaques (73). Although
arsenic does not cause point mutation, it does induce cell transformation, chro-
mosomal aberrations, sister chromatid exchanges, and gene amplification (2).
Arsenite can also initiate gene transcription by altering signal-transduction
molecules (2, 49). It is possible that arsenic induces atherosclerosis through its
actions on the structure and expression of related genes.
Nitrogen and phosphorus are important elements of DNA, RNA, and pro-
teins. Arsenic shares many similar chemical properties with these two elements
because they belong to the same group in the periodic table. Adenosine triphos-
phate (ATP) plays a major role in the regulation of many enzymes involving
the process of phosphorylation and dephosphorylation. Arsenic may hinder nor-
mal enzymatic functions by disrupting the formation of ATP from adenosine
diphosphate (ADP) and orthophosphate (49). As arsenite is known to react
strongly with sulfhydryl groups of proteins, it may also interfere with the nor-
mal biochemical functions of proteins regulated by the formation/destruction of
S S bonds involving the cysteine side chains in the proteins (49). Whether
arsenic may induce atherosclerosis through its interference with structural
or functional proteins involved in the atherosclerotic process requires further
investigations.
Oxidative stress has been known to promote atherosclerotic processes
through oxidation of low-density lipoprotein (LDL), activation of nuclear factor
κB, and induction of the immediate early genes c-myc and c-fos (2). A study
by Lynn and colleagues has shown that arsenite increases nicotinamide ade-
nine dinucleotide (NADH) oxidase activity and produces superoxide, which then
causes oxidative DNA damage in human vascular smooth muscle cells (34). Ad-
dition of the radical-scavenging enzyme superoxide dismutase also decreased
Blackfoot Disease and Arsenic 65
the frequency of arsenic-induced sister chromatid exchanges in human periph-
eral lymphocytes (74). A recent study carried out in Inner Mongolia, China
demonstrated well the presence of oxidative stress in human body with chronic
arsenic exposure (75). The study recruited 33 subjects exposed to drinking tube-
well water containing high arsenic concentration (mean value: 410 µg/L) for
about 18 years and a lower exposure group of 10 people (mean arsenic con-
centration of drinking water: 20 µg/L) as controls. Although the activity of
superoxide dismutase in the blood was not significantly different between both
groups, the serum level of lipid peroxides was significantly higher among the
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high-exposure group. Furthermore, the blood levels of inorganic arsenic and


the methylated metabolites correlated positively with serum level of lipid per-
oxide and negatively with blood levels of non-protein sulfhydryl, indicating an
induction of oxidative stress associated with chronic arsenic exposure and that
the methylated metabolites were associated with oxidative stress.
Atherosclerosis is characterized by apoptosis, and the rupture of the
atherosclerotic plaques abounding in apoptosis-induced necrotic debris is al-
ways associated with the onset of clinical cardiovascular events (2). Vascular
apoptosis is promoted by superoxide and oxidized LDL (76). Arsenic can pro-
duce a variety of stress responses leading eventually to apoptosis. Arsenic has
been found to induce apoptosis not only in hematopoietic malignant cells, but
also in solid tumors like neuroblastoma (37). It is also possible that arsenic can
produce apoptosis in the vasculature.
Some trace elements such as magnesium, copper, zinc, chromium and sele-
nium have been implicated as antiatherogenic and counteract the development
of cardiovascular disease (2). Arginine is the precursor of nitric oxide, a potent
endogenous vasodilator with the properties of inhibiting platelet aggregation,
monocyte adherence, chemotaxis, and vascular smooth muscle proliferation (2).
Substances with antioxidant effect such as α-tocopherol, ascorbic acid, and β-
carotene prevent oxidation of lipoprotein and thus protect the vessels from
atherosclerotic change (2). On the contrary, carbon monoxide, carbon disulfide,
certain aliphatic nitrates, lead, and cadmium have been implicated as athero-
genic and stimulate the development of cardiovascular disease (2). These ob-
servations suggest that the etiology of atherosclerosis is complex, and trace ele-
ments can play important roles during the formation of atherosclerotic plaques.
The co-contamination of arsenic and other trace elements from environ-
mental exposure could modulate the chronic toxicity of arsenic, and the inter-
action between arsenic and the other trace elements could also explain partly
the differential manifestation of its atherogenicity in different populations or
even among the same population in a single endemic area. Arsenic has been
shown to interact with several of these chemicals. Arsenic can lead to a signif-
icant increase in renal copper excretion and potentiate the effects of lead and
cadmium when used together (40). The effect of selenium can be antagonized
by administration of arsenic, probably through the action of enhancing biliary
66 C.-H. Tseng

excretion of selenium (41). Pershagen and colleagues reported that smoking


and arsenic inhalation have a multiplicative effect on lung cancer mortality in
smelter workers (42), and there are evidences of a positive interaction between
arsenic and benzo(a)pyrene, a carcinogen found as a by-product of combustion
(43). It is also observed that residents in the BFD area with lower serum con-
centrations of α- and β-carotene had a higher risk of developing ischemic heart
disease after the duration of arsenic exposure from the artesian well water was
considered (77). Arsenic has also been demonstrated to inhibit the absorption of
water, sodium, glucose, and leucine from the intestine of male Sprague Dawley
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rats in a dose-responsive pattern (78). Whether the absorption or the effect of


antioxidant substances such as α-tocopherol, ascorbic acid, and β-carotene can
also be inhibited by arsenic is not known.
Zinc is protective for the development of cardiovascular disease and has
been found to function in the control of apoptotic cell death by inhibiting the
disruption of endothelial cell integrity, DNA fragmentation, and cytolysis of
murine cells induced by tumor necrosis factor (22, 39). A simultaneous defi-
ciency of zinc as observed in some BFD patients can aggravate atherosclerosis
(22). Arsenic can compete with zinc in metal-binding proteins, displaying vici-
nal dithiols contained in zinc fingers of DNA binding and repair proteins and
transcription factors (22). The proteins’ conformational change can lead to al-
tered biologic function.
According to recent epidemiologic studies in the BFD areas, arsenic ex-
posure is also associated in a dose-responsive pattern with hypertension (45)
and diabetes mellitus (46–49), two of the major risk factors of atherosclero-
sis. Thus, it is possible that hypertension and diabetes mellitus explain partly
the higher rate of atherosclerotic disease associated with arsenic. However, the
atherosclerotic effect of arsenic is independent because the association found
in epidemiologic studies persists even after controlling the confounding effect
of both of these factors.
One question frequently asked is why endemic occurrence of PVD as ob-
served in the BFD areas in Taiwan is not similarly seen in other areas with
arsenic problems like Bangladesh, West Bengal, India, Inner Mongolia and
Guizhou of China and parts of United States, Mexico, Chile and Argentina.
Some of the explanations are the different manifestations of arsenic exposure
in different ethnicities, shorter durations of exposure in some areas, the effect
of different nutritional status and genetic difference in the metabolism of ar-
senic. However, the existence of other co-contaminants or other etiologic factors
cannot be completely excluded.

CAN HUMIC ACID BE CAUSALLY LINKED TO BFD?


Fluorescent compounds known as humic acids have also been suggested as a
cause of BFD (6). Lu demonstrated gangrene of the tails and feet in eight out
Blackfoot Disease and Arsenic 67
of sixteen mice after intraperitoneal injection of dissolved crystallized fluores-
cent compounds (79). However, there was no control mouse in that study, the
route of intraperitoneal injection would not occur in human beings, and the
experiment failed to report similar pathological changes as observed in BFD.
In a much larger series of 300 mice exposed to humic acids in their drinking
water for 24 months, no gangrenous changes were reported (80). The fluorescent
compounds seem to induce acute thrombosis via an action on blood coagulation
(81), which is quite different from BFD pathologically. As a matter of fact, humic
acids are a group of polymers with high molecular weights resulting from the de-
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composition of organic matter, particularly dead plants, they are widely spread
contaminants of water supplies and definitely not confined to the BFD areas.
Humic acids contain complex constituents with unstable physical and chemical
properties such as varying molecular weights. The measurement of fluorescent
intensity used in previous reports to quantify humic acids in water cannot
reflect the concentration of humic acids correctly. Ergotamine, D-lysergic acid
and/or ergometrine have been mentioned initially (82). However, these findings
have not been confirmed, and they were not mentioned again in more recent
studies. Furthermore, there is no known successful method to quantify humic
acids in human blood or tissues, whether humic acids with such large molecular
weights can be absorbed efficiently through gastrointestinal tract is not known
and the metabolism of humic acids remain unclear. Therefore, there has been
no epidemiologic evidence indicating the correlation between human exposure
to humic acids and circulatory diseases. Extrapolating the effects observed in
in vitro studies to humans can be very misleading. The association between hu-
mic acids and BFD, even if it could be demonstrated in an epidemiologic study,
could also be due to the confounding effect of the strong relation of BFD with
arsenic exposure. BFD patients has been shown to have hypercoagulability and
increased platelet aggregation (32) and to have a 30% less 6-keto-prostaglandin
F1α level and a normal level of thromboxane B2 (83). However, whether this is
due to an effect of humic acids or arsenic is not known. In recent studies, humic
acids have also been shown to induce the generation of nitric oxide in human
umbilical vein endothelial cells leading to cell injury (84), induce apoptosis (85),
and increase the adhesibility of neutrophils (86); however, all of these effects can
also be demonstrated with arsenic exposure. Humic acids have not been shown
to cause the dermatological or neurological defects, which are typically seen in
humans (19, 20) or animals (87, 88) exposed to arsenic. Because humic acids
are characterized by phenolic and phenolic carboxylic polymer structures con-
taining both –COOH and –OH as their main functional groups, they can form
complex with a variety of inorganic elements including arsenic (89). Whether
an interaction between humic acids and arsenic can play a role on the develop-
ment of BFD awaits further investigations. One of the possible interactions is
that humic acids may chelate large amounts of inorganic arsenic in the water
and release the arsenic in the gastrointestinal tract where it is absorbed.
68 C.-H. Tseng

Table 3: Comparison of the evidence of inorganic arsenic versus humic acids as a


cause of blackfoot disease.

Evidence Inorganic arsenic Humic acids


Epidemiologic Dose-responsive relationship Lacking
Studies demonstrated and similar
vascular disease observed in
other ethnicities exposed to
arsenic
Animal models Induction of atherosclerotic Intraperitoneal injection
plaques in sodium induced gangrene in
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arsenite-treated mice, mice demonstrated


compared to controls by Lu et al. (79), but
not confirmed in a
later study (80)
Gastrointestinal Inorganic arsenic can be Absorption and
absorption and absorbed efficiently and metabolic pathway
metabolism metabolic pathway is known unclear
Biological and A variety of properties compatible Induces thrombosis,
cellular studies with atherosclerosis (Table 2) neutrophil adhesion,
oxidative stress and
apoptosis

A comparison of the current evidences for arsenic and humic acids as po-
tential etiology of BFD is shown in Table 3.

CONCLUSIONS
The key features of BFD are summarized in Table 4. BFD in Taiwan can be
eradicated by cutting off the source of exposure (i.e., drinking artesian well

Table 4: Key features of blackfoot disease in Taiwan.

Features Remarks
Mean age of onset, years 52 (2–87)
Man:women 1.5:1
Hyperendemic areas Pei-Men, Hsueh-Chia, Pu-Tai and Yii-Chu
Peak incidence 1956–1960
Total cases (up to 1980s) >1,600
Prevalence in hyperendemic 6.51 to 18.81 per 1,000 population
areas in 1980s
Limb affected Mostly lower limbs, in rare cases fingers might be
involved
Clinical symptoms/signs Numbness, coldness, intermittent claudication,
absence of peripheral pulsation
Pathology 30% thromboangiitis obliterans and 70%
arteriosclerosis obliterans
Typical fate Ulceration, gangrene, spontaneous or surgical
amputation and death
Prognosis Annual death rate: 4.84 per 100 patient-years,
mean age at death: 62 years.
Major cause of death Cardiovascular disease
Blackfoot Disease and Arsenic 69
water), even the etiology of this devastating disease remains controversial. Al-
though arsenic is the most probable cause of BFD, the potential role of other
factors such as the humic acids cannot be completely excluded. The effects of
arsenic and humic acids may not be mutually exclusive and their interaction
on the promotion of BFD required further investigation. It has been a century
since the earlier cases of BFD were identified among residents of the south-
western coast of Taiwan. Science has solved the public health problems of BFD,
but more questions remain to be answered with regards to the real etiology and
the pathogenetic mechanisms of potential etiologic factors. Global problems of
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arsenic-contaminated drinking water are emerging (13, 90–92) rather than sub-
merging. Although the current devastating public health problems associated
with arsenic-contaminated drinking water in Bangladesh and West Bengal,
India can also be effectively tackled by the replacement of the contaminated
water with clean tap water, it may not be easy because of the wide geographical
distribution and the economic status of the countries. The story of BFD will
always be remembered and will not be ended in the foreseeable future.

ACKNOWLEDGEMENTS
The author thanks the National Science Council, Executive Yuan, Repub-
lic of China (NSC-86-2314-B-002-326; NSC-87-2314-B-002-245; NSC-88-2621-
B-002-030, NSC-89-2320-B-002-125 NSC-89-2318-B-002-007, NSC-90-2320-
B-002-197, NSC-92-2320-B-002-156 and NSC-93-2320-B-002-071), and the
Department of Health, Executive Yuan, Republic of China (DOH89-TD-1035)
for their support on the epidemiologic studies of blackfoot disease and diabetes
mellitus.

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