palacios, román, cifuentes biol trace element res (2012)

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Biological Trace Element Research

ISSN 0163-4984

Biol Trace Elem Res

DOI 10.1007/s12011-012-9355-3

Exposure to Low Level of Arsenic and Leadin Drinking Water from Antofagasta City

Induces Gender Differences in GlucoseHomeostasis in Rats

Javier Palacios, Domingo Roman &

Fredi Cifuentes


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Exposure to Low Level of Arsenic and Lead in Drinking

Water from Antofagasta City Induces Gender

Differences in Glucose Homeostasis in Rats

Javier Palacios & Domingo Roman & Fredi Cifuentes

Received: 8 December 2011 /Accepted: 2 February 2012# Springer Science+Business Media, LLC 2012

Abstract Populations chronically exposed to arsenic in

drinking water often have increased prevalence of diabetes

mellitus. The purpose of this study was to compare the glu-

cose homeostasis of male and female rats exposed to low

levels of heavy metals in drinking water. Treated groups were

SpragueDawley male and female rats exposed to drinking

water from Antofagasta city, with total arsenic of 30 ppb and

lead of 53 ppb for 3 months; control groups were exposed to

purified water by reverse osmosis. The two treated groups in

both males and females showed arsenic and lead in the hair of

rats. The -aminolevulinic acid dehydratase was used as a

sensitive biomarker of arsenic toxicity and lead. The activity

of -aminolevulinic acid dehydratase was reduced only in

treated male rats, compared to the control group. Treated

males showed a significantly sustained increase in blood

glucose and plasma insulin levels during oral glucose toler-

ance test compared to control group. The oral glucose toler-

ance test and the homeostasis model assessment of insulin

resistance demonstrated that male rats were insulin resistant,

and females remained sensitive to insulin after treatment. The

total cholesterol and LDL cholesterol increased in treated male

rats vs. the control, and triglyceride increased in treated female

rats vs. the control. The activity of intestinal Na+/glucose

cotransporter in male rats increased compared to female rats,

suggesting a significant increase in intestinal glucose absorp-

tion. The findings indicate that exposure to low levels of

arsenic and lead in drinking water could cause gender differ-

ences in insulin resistance.

Keywords Gender differences . Arsenic . Lead . Drinking

water. Insulin resistance . Rat

Introduction

The prevalence of diabetes mellitus is increasing in Chilean

population, similar to the USA, Canada, Argentina, and

Uruguay [1]. The association between chronic exposure to

inorganic arsenic at high levels (>100 ppb) and diabetes

mellitus was confirmed by several epidemiological studies

in Taiwan, Bangladesh, and Mexico [24]. The low-level

lead exposure is associated with hypertension, cognitive

dysfunction, neurobehavioral disorders, and renal impair-

ment [5], but not with type 2 diabetes mellitus.

Chronic arsenic exposure via drinking water has been

reported in population of Antofagasta (latitude 23 38 S,

longitude 70 24 W). Since September 2004, the total

arsenic level in drinking water from Antofagasta City has

approximately 40 [6] and 20 ppb [7]. The World Health

Organization set 10 ppb as the recommended limit for

arsenic in drinking water [8]. Unfortunately, there are no

studies that show the toxic effects of lead in drinking water

from Antofagasta city, only a few studies on the toxic effect

of lead in salt rivers and in soil [9, 10].

J. Palacios (*)

Departamento de Qumica, Universidad Catlica del Norte,

Angamos 0610,

Antofagasta, Chile

e-mail: [email protected]

D. Roman

Laboratorio de Qumica Bio-Inorgnica y Analtica Ambiental,

Departamento de Qumica, Universidad de Antofagasta,

Angamos 601,

Antofagasta, Chile

F. Cifuentes

Experimental Physiology Laboratory (EPhyL),

Departamento Biomdico, Universidad de Antofagasta,

Angamos 601,

Antofagasta, Chile

Biol Trace Elem Res

DOI 10.1007/s12011-012-9355-3


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Arsenic exposure can potentially induce type 2 diabetes

mellitus by: homeostasis alteration of glucose, inhibition of

glucose uptake by adipocytes and skeletal muscle [11, 12],

or interference with the glucose metabolism in the liver [13,

14]. Other mechanisms that could be involved in glucose

homeostasis are the Na+/glucose cotransporter and the ac-

tivity of incretin in the small intestine. It is known that the

Na+/glucose cotransporter activity is increased by diabetesmellitus and is regulated by insulin [15], but there are few

studies on the effect of heavy metals on Na+/glucose

cotransporter.

Because Na+/glucose cotransporter (SGLT1; SLC5A1) is

the primary glucose transporter involved in intestinal ab-

sorption of mammals [16], we studied this in rats. This

cotransporter is located in the apical membrane of small

intestinal brush, which has a 2:1 stoichiometry (sodium/

glucose; Km01050 M), and is inhibited by phloridzin,

Ki0510 M [17].

The purpose of this study was to compare the insulin

sensitivity and glucose homeostasis of male and female ratsexposed to low levels of arsenic and lead in drinking water.

Materials and Methods

Drugs

The following drugs were used in this study: thiopental

sodium (Sigma, St. Louis, MO), -aminolevulinic acid

(Merck, Germany), HgCl2 (Merck, Germany), 4-(dimethy-

lamino) benzaldehyde (Merck, Germany), sodium arsenite

(Merck, Germany), and phloridzin (Sigma, USA). The

drugs were dissolved in distilled deionized water.

Animals

SpragueDawley rats, male and female (1516 weeks of

age, 300450 g), from the breeding colony at the Anto-

fagasta University were used. All rats were housed in

groups of two or three in a temperature-controlled, light-

cycled (08002000 hours) room with ad libitum access

to drinking water and standard rat chow (Champion,

Santiago). The investigation conformed to the Guide for

the Care and Use of Laboratory Animals published by

the U.S. National Institutes of Health (NIH Publication

No. 8523, revised 1996), and the local animal research

committee approved the experimental procedure used in

the present study.

Male and female rats were randomized into four groups

(six to ten animals each): treated male rats, control male

rats, treated female rats, and control female rats. Treated

groups drank drinking water from Antofagasta city

(30 ppb As and 53 ppb Pb), or control groups drank

purified water by reverse osmosis (3 ppb As and


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The Oral Glucose Tolerance Test and Plasma Insulin

Animals were fasted 12 h prior to administration of the oral

glucose tolerance test (OGTT). Samples of whole blood

were collected from a lateral tail incision in male and female

rats slightly anesthetized (dose of 20 mg/kg body weight

with thiopental), as recommended by some authors [20, 21].

A level of glucose (2 g/kg body weight) via oral was used aspreviously described [12]. Glycemia was measured in the

tail of rats every 15 min and for 90 min using OneTouch

Ultra Blood Glucose System; in the same blood sample was

also determined plasma insulin level by RIA.

Blood -Aminolevulinic Acid Dehydratase

The -aminolevulinic acid dehydratase (ALA-D) activity of

erythrocyte is a sensitive biomarker for arsenic and lead

toxicity [22, 23]. The method is based on the conversion

of-aminolevulinic acid by the enzyme to porphobilinogen.

The activity of blood ALA-D was assayed according toBerlin and Schaller procedure [24]. Total volume of

0.2 mL of heparinized blood was mixed with 1.3 mL of

distilled water and incubated for 10 min at 37C until

complete hemolysis. The standard -aminolevulinic acid

(0.01 M; 1 mL) was added to the samples, and then, the

tubes were incubated for 60 min at 37C. Porphobilinogen

formation is linear for at least 2 h at 37C.

The enzyme activity was stopped after 1 h by adding

1 mL of HgCl2 (1.35 g per 100 mL of 10% trichloroacetic

acid). The reaction mixture was centrifuged (3,000 rpm

10 min). An equal volume of Ehrlich reagent was added to

the supernatant, and the absorbance was recorded at 555 nm

after 5 min. The ALA-D activity was calculated by absor-

bance and hematocrit according to this relationship: ALA Dactivity Abs 100 2 35=Ht 60 0:062; where 20conversion factor from -aminolevulinic acid to porphobili-

nogen, 350dilution factor, 60 min0incubation time, and

0.062 L mol1 cm1 is the extinction coefficient of

porphobilinogen.

Activity of Intestinal Na+/Glucose Cotransporter

(SGLT1; SLC5A1)

The rats were anesthetized by intraperitoneal administration

of thiopental at a dose of 60 mg/kg body weight followed by

a midline laparotomy. Briefly, the protocol used was from

Diez et al. [25]. A segment of about 20 cm of proximal

duodenum was perfused using two catheters. The intestinal

content was washed out with physiological solution at 37C.

After a period of equilibrium (10 min), a bolus of 1 g

glucose in 1.5 ml 0.9% NaCl was infused within 1 min.

Cotransporter SGLT1 activity was calculated as the differ-

ence between the concentration of glucose eluated, in the

presence or absence of 0.5 mM phloridzin (a specific com-

petitive inhibitor of SGLT1), regarding the length of the

segment of the small intestine and 10 min of absorption.

Statistical Analysis

All results were evaluated by ANOVA with Dunnett test to

detect significant differences. Area under the curve (AUC)glucose and insulin values were calculated using the trapezoi-

dal rule. All data are expressed as the meansstandard error of

the mean, and p


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the treated female rats (0.540.03; p


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significant gender difference in the insulin AUC. The insulin

AUC was significantly higher in the treated male rats

(80.3 ngmL1min1 control group versus 231.4 ng

mL1min1 treated group;p


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control group, whereas there were no significant differences

in ALA-D activity of the female rats. These data suggest that

the toxic effect was mainly in the male rats than in the

females.

It has been proposed that men may be more affected by

the toxic effects of arsenic than women [33]. This hypoth-

esis is supported by the methylation of inorganic arsenic

which is considered a detoxification pathway [34]. Actually,women have a greater proportion of methylated metabolites

of arsenic compared to men, which would decrease arsenic

toxicity in the body [35, 36]. However, the theory of the

methylation of inorganic arsenic as a detoxification process

has been revised [37]; in fact, it was demonstrated that other

trivalent methylated species have high toxicity [38]. There-

fore, further investigation is required to get a deeper knowl-

edge of detoxification mechanisms in gender differences.

An important finding was that exposure to drinking water

from Antofagasta city caused gender differences in glucose

homeostasis. Indeed, in the treated groups only the male rats

were insulin resistant, as indicated by measurements ofglucose and insulin levels in blood during OGTT. The male

rats showed a significantly sustained increase in blood glu-

cose and plasma insulin levels during OGTT compared to

the control group. In addition, HOMA-IR confirmed that

insulin resistance was presented only in the male rats. In

contrast to insulin resistance observed above, the treated

female rats were more sensitive to insulin during OGTT.

The lipid profile was associated with insulin resistance in

male rats. In fact, total cholesterol and LDL cholesterol

increased in the treated males vs. the control rats, while

glucose tolerance and insulin sensitivity decreased. These

results suggest that lipid intake or lipogenesis exceeds the

storage capacity of the tissues, leading to glucose intolerance

and insulin resistance in the treated male rats. However, in the

treated female rats, triglyceride level increased, and glucose

tolerance and insulin sensitivity did not change. This is con-

sistent with improved glucose tolerance and insulin sensitivity

in the female rats under excessive lipid metabolism compared

with the male rats [39].

Another gender difference between the two groups after

treatment was in the intestinal absorption of glucose. The

activity of intestinal SGLT1 in the male rats increased com-

pared with the females. In addition, the slope of the curve of

glycemia during the first 30 min of OGTT was significantly

higher in the treated male rats vs. the treated females.

Therefore, the increase in intestinal glucose absorption

may be another factor that enhances the postprandial blood

glucose level in treated male rats.

It is known that intestinal SGLT1 activity is inhibited by

insulin in normal rats [15]. However, Fujica et al. showed that

an increase in intestinal glucose absorption by SGLT1 cotrans-

porter is associated with postprandial hyperglycemia before

the onset of insulin resistance and hyperinsulinemia in obese

type 2 diabetic rats [40, 41]. Actually, we may hypothesize

that intestinal SGLT1 activity was not inhibited by the high

level of plasma insulin observed in the treated groups during

OGTT. In fact, preincubation with insulin in a segment of

intestine did not inhibit the SGLT1 activity in the treated male

rats compared to the control group (data not shown).

Although the purpose of this paper was not to examine

whether insulin resistance was due to arsenic or lead, orboth, we repeated OGTT in the male rats treated with 30 ppb

sodium arsenite in purified water. The data indicated that

arsenic (sodium arsenite) produced insulin resistance but

less than in male rats exposed to drinking water. Izquierdo-

Vega showed that exposure to 1.7 ppm sodium arsenite pro-

duced insulin resistance in the male rats [42]; Paul et al. found

that 50 ppm sodium arsenite caused insulin resistance in mice

[43]; glucose metabolism is altered by exposure to 50 ppb

sodium arsenite in drinking water in mice [44]. Lin et al.

showed that exposure to low levels of lead accelerates pro-

gressive renal insufficiency in patients with chronic renal

disease (without diabetes) [28]. We think the lead could en-hance resistance to insulin, but more experiments are required

to fully understand this point.

In conclusion, exposure to low level of arsenic and lead

in drinking water of Antofagasta city alters the sensitivity to

insulin in a gender-dependent way. The role of intestinal

SGLT1 activity in the insulin resistance of male rats is

relevant probably because it contributes to the increase of

intestinal glucose absorption [45].

Acknowledgments We would like to thank Blanca Alvarez Carvajal

from Laboratorio Clnico Hormonal RadioLab, Laboratorio Clnico

Blanco for the technical assistance in this study. This work was in part

supported by grants from Fondo Interno de Investigacin Cientfica de

la Universidad Catlica del Norte (DGIP 220203-10301206) and

Direccin General de Investigacin (DIRINV 1339-2007) de la Uni-

versidad de Antofagasta.

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