Arsenic-induced dyslipidemia in male albino rats: comparison between trivalent and pentavalent inorganic arsenic in drinking water

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1 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 DOI /s z RESEARCH ARTICLE Arseni-indued dyslipidemi in mle lino rts: omprison etween trivlent nd pentvlent inorgni rseni in drinking wter Open Aess Olusegun K. Afoli 1,3, Adedoj D. Wusu 1,, Olisi O. Ogunrinol 1,, Esther O. Am 1,6, Dvid O. Byemi 1, Oluwtosin. A. Dosumu 1, Okehukwu. B. Onunkwor 1, Elizeth. A. Blogun 1,5, Olusegun O. Odukoy nd Oldipo Ademuyiw 1* Astrt Bkground: Reent epidemiologil evidenes indite lose ssoition etween inorgni rseni exposure vi drinking wter nd rdiovsulr diseses. However, the ext mehnism of this rseni-medited inrese in rdiovsulr risk ftors remins enigmti. Methods: In order to investigte the effets of inorgni rseni exposure on lipid metolism, mle lino rts were exposed to 5, 1 nd 15 ppm rseni s sodium rsenite nd 1, 15 nd ppm rseni s sodium rsente respetively in their drinking wter for 1 weeks. Results: Dyslipidemi indued y the two rsenils exhiited different ptterns. Hypoholesterolemi hrterised the effet of rsenite t ll the doses, ut rsente indued hyperholesterolemi t the 15 ppm As dose. Hypertriglyeridemi ws the hllmrk of rsente effet wheres plsm free ftty ids (FFAs) ws inresed y the two rsenils. Reverse holesterol trnsport ws inhiited y the two rsenils s evidened y deresed HDL holesterol onentrtions wheres hepti holesterol ws inresed y rsenite (1 ppm As), ut deresed y rsenite (15 ppm As) nd rsente (1 ppm As) respetively. Brin holesterol nd triglyeride were deresed y the two rsenils; rsente deresed the renl ontent of holesterol, ut inresed renl ontent of triglyeride. Arsenite, on the other hnd, inresed the renl ontents of the two lipids. The two rsenils indued phospholipidosis in the spleen. Arsenite (15 ppm As) nd rsente (1 ppm As) inhiited hepti HMG CoA redutse. At other doses of the two rsenils, hepti tivity of the enzyme ws up-regulted. The two rsenils however up-regulted the tivity of the rin enzyme. We oserved positive ssoitions etween tissue rseni levels nd plsm FFA nd negtive ssoitions etween tissue rseni levels nd HDL holesterol. Conlusion: Our findings indite tht even though su-hroni exposure to rsenite nd rsente through drinking wter produed different ptterns of dyslipidemi, our study identified two ommon denomintors of dyslipidemi nmely: inhiition of reverse holesterol trnsport nd inrese in plsm FFA. These two denomintors (in ddition to other individul perturtions of lipid metolism indued y eh rsenil), suggest tht in ontrst to strengthening dose-dependent effet phenomenon, the two forms of inorgni rseni indued lipotoxi nd non-lipotoxi dyslipidemi t low or medium doses nd these might e responsile for the rdiovsulr nd other disese endpoints of inorgni rseni exposure through drinking wter. Keywords: Trivlent inorgni rseni, Pentvlent inorgni rseni, Drinking wter, Dyslipidemi * Correspondene: deld@yhoo.om 1 Deprtment of Biohemistry, Federl University of Agriulture, Aeokut, Nigeri Full list of uthor informtion is ville t the end of the rtile 15 Afoli et l. This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly redited. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge of 15 Bkground Among the plethor of toxints, rseni rnks s n environmentlly uiquitous nd epidemiologilly importnt metlloid urrently poisoning tens of millions of people worldwide [1, ]. It is found in oth inorgni nd orgni forms nd in different vlene or oxidtion sttes in the environment. Chroni exposure to rseni is ssoited with wide rnge of toxi effets [1, 3]. Cner of the skin, lung, kidney, liver nd urinry ldder re the importnt ners ssoited with these toxi effets [1 3]. Among the non-ner effets of rseni, dietes mellitus, goitre, heptomegly, ronhitis, ronhietsis, ererovsulr nd rdiovsulr diseses, re well doumented [3 5]. Epidemiologil studies hve demonstrted tht ingestion of rseni through drinking wter might e responsile for these rinogeni nd non-ner effets of rseni [3,, 6]. In spite of this lrge ody of informtion out the toxi effets of rseni, the preise mehnisms of tion for the mny disese endpoints following ute nd hroni rseni exposure, s well s the threshold for iologi effets nd disese risks, remin enigmti [1]. Over the yers, epidemiologil studies hve identified lipid nd lipoprotein normlities s independent risk ftors in the pthogenesis nd progression of theroslerosis nd rdiovsulr diseses [7, 8]. There is lso inresing evidene tht environmentl ftors/ontminnts (most espeilly hevy metls) ontriute to this dyslipidemi [7, 9, 1]. Studies onduted in rseniexposed popultions reveled prevlene of nerly full spetrum of rdiovsulr diseses inluding hypertension, theroslerosis, lkfoot disese, ishemi hert diseses, et. [3, ]. Therefore, it seems resonle to hypothesise tht the ssoition etween rseniosis nd rdiovsulr diseses my e medited through modifition of lipid nd lipoprotein metolism. The work reported here explored this hypothesis. Mterils nd methods Chemils Sodium rsenite nd sodium rsente were produts of Sigm-Aldrih, Missouri, USA. Animls nd tretment Experimentl protools were onduted in ord with guidelines of the Institutionl Animl Cre nd Use Committee nd were pproved y the Animl Ethil Committee of the Deprtment of Biohemistry, Federl University of Agriulture, Aeokut, Nigeri. Fifty-six mle Wistr rts (red in the College of Veterinry Mediine, Federl University of Agriulture, Aeokut, Nigeri) with men ody weight of 13 g were used for the experiments. They were housed in n niml room with norml ontrolled temperture ( ± C) nd regulr 1 h light drk yle (6: 18: h). They were llowed 1 dys to limtise efore the ommenement of rseni exposure. The nimls were mintined on stndrd pellet diet. Animls were divided into 8 groups of 7 nimls eh. While groups served s ontrol nd reeived distilled wter, the remining groups (3 groups eh) were exposed to 5, 1 nd 15 ppm rseni s sodium rsenite nd 1, 15 nd ppm rseni s sodium rsente respetively in their drinking wter for 1 weeks. These rseni onentrtions were hosen sed on previous studies [11 15]. At the end of rseni exposure, lood ws olleted from the nimls into heprinised tues y rdi punture under light ether nesthesi fter n overnight fst. Liver, kidney, hert, lung, rin nd spleen were lso removed from the nimls for rseni nd lipid nlyses. An liquot of the lood smples ws tken for rseni determintion while the remining lood smples were entrifuged to seprte plsm nd red lood ells. All smples were stored t C until nlysed. Arseni determintion A portion of the frozen orgns ( mg) nd whole lood (. ml) were digested in nitri id. Totl rseni (whih would inlude inorgni nd orgni forms) ws determined using tomi sorption spetrometry. Results re expressed s μg As/ml for lood nd μg As/gwet weight for the orgns. Biohemil nlyses Plsm nd lipoprotein lipid profiles Determintion of the mjor lipids (holesterol, triglyerides, phospholipids nd free ftty ids) in plsm nd lipoproteins followed estlished proedures. Detils of these hve een given in our erlier studies [7, 16 19]. Orgn nd erythroyte lipid profiles Lipids were extrted from the orgns (liver, kidney, hert, lung, rin nd spleen) s desried y Folh et l. (1957) [] while extrtion of erythroyte lipids followed the proedure desried y Rose nd Oklnder (1965) [1]. After wshing with.5 M KCl solution, liquots of the lipid extrts were then used for the determintion of lipid profiles. Detils of these re given s reported erlier [7, 16 19]. Determintion of hepti nd rin HMG-CoA redutse tivity This ws determined ording to the method of Ro nd Rmkrishnn (1975) [] y mesuring the hepti nd rin onentrtions of HMG-CoA nd mevlonte. The rtio of HMG-CoA to mevlonte is tken s n

3 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 3 of 15 index of the tivity of HMG-CoA redutse. An inrese in this rtio indites inhiition of holesterogenesis while derese indites enhned holesterogenesis. Sttistil nlysis Results re expressed s men ± SEM. One wy nlysis of vrine (ANOVA) followed y Tukey s test ws used to nlyze the results with p <.5 onsidered signifint. Assoitions mong the prmeters nd their mgnitudes were determined using Person orreltion. Results Tle 1 depits the index of rseni exposure (expressed s weekly rseni intke/rt) lulted y the formul: wter onsumption (ml) rseni onentrtion (ppm). Arseni intke per week ws reltively onstnt in eh dose group. For the rsenite-exposed rts, eh niml ingested 7.1 ±.9 mg As/week in the 5 ppm group, while rseni ingestion ws ±.69 mg As/week nd ±.6 mg As/week in the 1 ppm nd 15 ppm groups respetively. For the 1 weeks of exposure, eh niml in the 5 ppm group ingested totl of mg of rseni, while n niml in the 1 ppm group nd 15 ppm group hd totl rseni intke of mg nd 7.9 mg respetively. The rsenteexposed nimls lso showed onstnt rte of rseni intke. In the 1 ppm group, n niml onsumed on the verge, totl of 1.6 ±.68 mg As/week, while in the 15 ppm nd ppm groups, the intke per week were 16. ± 1.7 mg nd. ± 1.87 mg respetively. Totl rseni intke in the groups mounted to 1.75 mg, 19. mg nd 6. mg per rt for the 1 ppm, 15 ppm nd ppm doses respetively. Arseni levels in some of the tissues in rts dministered either rsenite or rsente in their drinking wter re shown in Tle. Although the dministrtion of the two rsenils resulted in n umultion of rseni in lood, liver, kidney, hert, lung, rin nd spleen of the nimls, only in the kidney of rsente-treted nd the rin of rsenite-treted nimls ws the umultion found to e dose-dependent. In rsenite-treted nimls, exposure to n rseni dose of 5 ppm resulted in rseni ontent of 35 μg As/ml of lood. Douling of the rseni dose did not result in ny further inrese in rseni ontent. Rther, rseni ontent of the lood pprohed sturtion. At the highest dose of rseni in the sodium rsenite group (15 ppm), rseni ontent of the lood redued from 31 μg As/ml to 58 μg As/ml. In the rsente-treted nimls, exposure to n rseni dose of 1 ppm resulted in n rseni ontent of 8. μg As/ml of lood. Inresing the rseni dose resulted in further inrese in rseni ontent from 8. μg As/ml to 67.1 μg As/ml. Inresing the rseni dose to ppm did not result in ny ppreile inrese in rseni ontent of lood. Rther, rseni ontent of lood in this group of nimls peked t 73.3 μg As/ml. In rsenite-exposed nimls, the spleen umulted the highest mount of rseni, wheres in rsente-exposed nimls, kidney umulted the highest mount of rseni. Dyslipidemi indued y rsenite ws hrterised y hypoholesterolemi t ll the rseni doses (Fig. 1). At Tle 1 Weekly nd totl rseni intkes of eh niml on exposure to sodium rsenite nd sodium rsente for 1 weeks through drinking wter Week Sodium rsenite Sodium rsente Arseni Dose Arseni Dose 5 ppm 1 ppm 15 ppm 1 ppm 15 ppm ppm Totl intke (mg) Men intke/week (mg) (± S.E.M) 7.1 ± ± ± ± ± 1.7. ± 1.87

4 Tle Arseni onentrtions in the tissues of the nimls on exposure to sodium rsenite nd sodium rsente for 1 weeks through drinking wter Sodium rsenite Sodium rsente Arseni dose Arseni dose Tissue Control 5 ppm 1 ppm 15 ppm Control 1 ppm 15 ppm ppm Blood (μg/ml) 11. ± ± ± ± ± ± ± ± 1.33 Liver (μg/g wet weight) 1.73 ± ± ± 1.7. ± ± ± ± ±.96d Kidney (μg/g wet weight).6 ± ± ± ±.3d.6 ± ± ± ±.99d Brin (μg/g wet weight).69 ± ±..88 ± ±.33d 1.7 ± ± ± ±.1d Hert (μg/g wet weight). ± ± ± ±.3. ±.3.16 ± ± ±.99 Lung (μg/g wet weight).71 ±.3.97 ± ± ± ±. 6.6 ±.88.8 ± ±.6 Spleen (μg/g wet weight) 3.37 ±.9.35 ± ± ± ± ± ± ± 1.6 Eh vlue represents the men ± S.E.M. of 7 rts. Vlues within row with different lphets for eh rseni ompound re signifintly different t p <.5 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge of 15

5 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 5 of 15 Plsm Plsm 1 HDL 1 HDL Cholesterol Conentrtion (mg/dl) 8 6 Cholesterol Conentrtion (mg/dl) 8 6 d d Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 1 Effets of rsenite nd rsente on plsm nd HDL holesterol onen-trtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5 oth 1 ppm nd 15 ppm doses of rseni in rsenitetreted nimls, holesterol redued from 56.8 ± 3.1 mg/ dl in ontrol to 1.1 ± 3.35 mg/dl nd 3.9 ± 3.87 mg/dl respetively. Arsente, on the other hnd, inresed plsm holesterol y 69 % t the 15 ppm As dose ut deresed this lipid y 3 % t 1 ppm As. HDL holesterol ws redued to the sme extent y the two rsenils. At the highest dose of eh rsenil, HDL holesterol ws just out 5 % of ontrol vlues (Fig. 1). As indited in Fig., rsenite eliited hormeti response in plsm triglyeride onentrtion in whih the 5 ppm As dose up-regulted wheres the 1 ppm As dose down-regulted the plsm triglyeride ontent. The 5 ppm rseni dose in rsenite-exposed nimls inresed the plsm triglyeride ontent from 6.81 ±.5 to 55.3 ± 3.33 mg/dl. Douling the rseni dose resulted in 5 % redution in triglyeride wheres plsm triglyeride level t highest rseni dose ws not signifintly different from the ontrol (p >.5). Arsente, on the other hnd, indued hypertriglyeridemi t ll the doses of rseni employed. Arsenite exposure t the 5 ppm As dose lso used n elevtion of the HDL triglyeride ontent while, t 15 ppm As dose, the lipid ws signifintly redued (p <.5). With rsente exposure, however, there ws no signifint hnge in HDL triglyeride onentrtions exept t the 15 ppm As dose where n inrese in onentrtion ws oserved. Plsm phospholipid onentrtions were signifintly redued in oth rsenite- nd rsente-exposed rts t the highest dose (p <.5) while no signifint hnges t oth the low nd medium doses were oserved (Fig. 3). Exposure of the nimls to either rsenite or rsente did not result in ny signifint hnges in the HDL phospholipid ontents t ll the doses employed, lthough there ws grdul depression of the onentrtions s the rsente doses were inresed (Fig. 3). Cholesterol onentrtion in the LDL + VLDL frtion ws inresed on exposure to rsenite nd rsente t ll rseni doses (Fig. ). The inrese in the rseniteexposed rts mounted to 9 % wheres in rsenteexposed rts the inreses mounted to 3 %, 165 % nd 99 % in the 1 ppm, 15 ppm nd ppm groups respetively. Triglyeride ontents of the LDL + VLDL frtion were not ffeted y exposure to rsenite ut rsente used -fold inrese in sme lipid t 15 ppm nd ppm As doses (Fig. ). The men phospholipid onentrtions in LDL + VLDL s depited in Fig. indite tht oth rsenite nd rsente exposures resulted in depletion of LDL + VLDL phospholipids. In rsenite-exposed nimls, the highest depletion of 5 % ws oserved t the 1 ppm As dose, ut with rsente, the redution ws mximl in the highest dose group ( ppm) where 1 % redution ws oserved. The erythroyte lipid ontents of the nimls re shown in Fig. 5. Both rsenite nd rsente exposures resulted in signifint depression of the holesterol ontents of the red lood ells in the nimls (p <.5). Arsenite-exposure resulted in 35 % nd % dereses in holesterol ontents of the erythroyte of rts with 1 ppm nd 15 ppm As respetively. In rsenteexposed rts, 3, nd 39 % redution resulted from exposure to 1 ppm, 15 ppm nd ppm rseni doses respetively. Arsenite exposure did not ffet triglyeride ontents of rt erythroytes s no signifint

6 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 6 of Plsm HDL 15 Plsm HDL Triglyeride Conentrtion (mg/dl) 1 5 Triglyeride Conentrtion (mg/dl) 1 5 Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig Effets of rsenite nd rsente on plsm nd HDL triglyeride onen-trtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5 hnge (p >.5) ws oserved in the onentrtions. Exposure to 1 ppm As in rsente-exposed nimls, however, resulted in 31 % inrese in erythroyte triglyeride onentrtion (p <.5). Inrese in the rseni dose did not result in ny further signifint inrese in the erythroyte triglyeride. Phospholipid onentrtions in the erythroyte were mrkedly lowered on exposure to the two rsenils, lthough in oth ses, the redution ws not dosedependent. While the redution mounted to 51, 9 nd 39 % respetively in rsenite-treted nimls, it mounted to 3, 16 nd 9 % respetively in rsente-treted nimls. Plsm nd erythroyte FFA onentrtions re depited in Fig. 6. In oth rsenite- nd rsente-exposed nimls, there ws grdul inrese in oth plsm nd erythroyte FFA onentrtions s the rseni doses were inresed. Comprtively, in the plsm, the inrese ws more pronouned in rsente-treted nimls wheres in the erythroytes the inrese ws more pronouned in rsenite-treted nimls. The effets of the rsenils on hepti, renl nd rin holesterol onentrtions re depited in Fig. 7. While rsenite inresed hepti nd renl holesterol y 35 nd 8 % respetively, it deresed rin holesterol y s muh s 5 % t the highest dose of rseni. Arsente, on the other hnd, inresed hepti holesterol ut deresed renl nd rin holesterol onentrtions y 5 nd 31 % respetively. 8 Plsm HDL 8 Plsm HDL Phospholipid Conentrtion (mg/dl) 6 Phospholipid Conentrtion (mg/dl) 6 Control 5 ppm 1 ppm 15 ppm Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsenite) Arseni Dose (Sodium rsente) Fig. 3 Effets of rsenite nd rsente on plsm nd HDL phospholipid onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5

7 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 7 of 15 LDL+VLDL Lipid Conentrtions (mg/dl) Cholesterol Triglyeride Phospholipid LDL+VLDL Lipid Conentrtions (mg/dl) 1 Cholesterol Triglyeride Phospholipid 8 6 Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. Effets of rsenite nd rsente on LDL + VLDL holesterol, triglyeride nd phospholipid onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5 Crdi, pulmonry nd spleni holesterol ontents re shown in Fig. 8. Exposure to the two rsenils resulted in the depletion of the rdi holesterol while inresing pulmonry nd spleni holesterol ontents. At the highest dose of eh rsenil, rdi holesterol ws just 3 % (rsenite) nd 5 % (rsente) of ontrol vlues respetively. In the pulmonry omprtment, the pttern of inrese ws similr for the two rsenils. In the spleni omprtment, however, while the highest inrese ( %) ws oserved with the highest dose of rseni in rsenite-exposed nimls, it ws the 1 ppm As in rsente-exposed nimls tht eliited the highest inrese (1 %). Arsenite eliited hormeti response in hepti triglyeride onentrtions (Fig. 9). While the 5 ppm rseni dose up-regulted triglyeride onentrtion y 35 %, the highest dose of rseni (15 ppm) lso used 35 % redution in triglyeride onentrtion (Fig. 9). Arsente, on the other hnd, inresed hepti triglyeride (11 % nd 3 %) t 15 nd ppm As doses respetively. Both rsenite nd rsente inresed the renl ontents of triglyeride. The inrese ws more pronouned in rsenite-treted nimls. The two rsenils lso deresed to the sme extent the triglyeride onentrtion of the rin, lthough the derese ws not dose-dependent. 5 Cholesterol Triglyeride Phospholipid 5 Cholesterol Triglyeride Phospholipid Erythroyte Lipid Conentrtions (mg/dl) Erythroyte Lipid Conentrtions (mg/dl) Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 5 Effets of rsenite nd rsente on erythroyte holesterol, triglyeride nd phospholipid onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5

8 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 8 of 15 6 Plsm Erythroyte 5 Plsm Erythroyte d Free Ftty Aid Conentrtion (mg/dl) Free Ftty Aid Conentrtion (mg/dl) 3 1 Control 5 ppm 1 ppm 15 ppm Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsenite) Arseni Dose (Sodium rsente) Fig. 6 Effets of rsenite nd rsente on plsm nd erythroyte free ftty id onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5 Crdi, pulmonry nd spleni triglyeride ontents re shown in Fig. 1. The hert responded to the presene of the two rsenils in similr pttern. While the hert responded to the lowest dose of eh rsenil with derese (6 % in rsenite nd 8 % in rsente), triglyeride ontents egn to inrese with eh inresing dose of the rsenils, lthough the inrese did not reh sttistil signifine (p >.5) within the exposed groups. In ontrst to the hert, there ws dosedependent derese in the triglyeride ontents in the lungs in rsente exposure. Although rsenite lso inresed pulmonry triglyerides, the inrese ws not dose-dependent. Exposure to the two rsenils did not result in ny signifint hnge in spleni triglyeride. The men phospholipid onentrtions in the tissues of the nimls s depited in Figs. 11 nd 1 indite tht exposure to oth rsenils resulted in spleni phospholipidosis. Arsenite exposure lone resulted in rin phospholipidosis while rsente exposure t the highest dose ( ppm As) resulted in hepti phospholipidosis. In ontrst however, rsente used signifint redution in phospholipid onentrtions in the rin, hert nd lung of the nimls (Fig. 11) wheres it ws only in the hert, liver nd lung tht phospholipid ws redued on exposure to rsenite (Figs. 11 nd 1). Renl phospholipids were not ffeted y ny of the rsenils irrespetive of the dose (Fig. 11). 15 Liver Kidney Brin 15 Liver Kidney Brin Cholesterol Conentrtions (mg/g tissue weight) 1 5 Cholesterol Conentrtions (mg/g tissue weight) 1 5 Control 5 ppm 1 ppm 15 ppm Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsenite) Arseni Dose (Sodium rsente) Fig. 7 Effets of rsenite nd rsente on hepti, renl nd rin holesterol onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5

9 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 9 of 15 Cholesterol Conentrtions (mg/g tissue weight) 5 Hert Lung Spleen 3 1 Cholesterol Conentrtions (mg/g tissue weight) Hert Lung Spleen Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 8 Effets of rsenite nd rsente on hert, lung nd spleen holesterol onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5 As indited in Fig. 13, rsenite (15 ppm As) nd rsente (1 ppm As), inhiited hepti HMG CoA redutse tivity. At other doses of the rsenils, hepti HMG CoA redutse ws up-regulted y 17 % y the two rsenils respetively. The tivity of the rin enzyme ws lso up-regulted y the two rsenils (rsenite 56 % nd rsente 5 %). The ssoitions etween tissue rseni levels nd some lipid prmeters re depited in Tle 3. While tissue rseni levels orrelted positively with plsm FFA, reverse holesterol trnsport ws negtively orrelted with tissue rseni levels. Tissue rseni levels (with the exeption of hepti rseni vs. hepti HMG CoA redutse nd rin rseni vs. hepti HMG CoA redutse), lso orrelted positively with hepti nd rin HMG CoA redutse. Disussion Arseni umulted in ll the orgns investigted irrespetive of the hemil speies dministered with onentrtions rised y mny folds ompred to ontrol. The rther high level of rseni in the lood of the exposed rts hs een reported y others [13, 3] nd is minly ontriuted y the erythroytes whih re responsile for the distriution of rseni throughout the ody. Following exposure to inorgni rseni, As V is sequentilly redued to As III. The As III produed y this redution or from diret ingestion is then oxidtively methylted to give pentvlent orgni rsenils, monomethylrsoni id (MMA) nd dimethylrsini id (DMA) []. This in turn is funtion of ellulr uptke of the inorgni rseni in vrious tissues [5]. Uptke of sustnes in ell is funtion of the ell memrnes. Triglyeride Conentrtions (mg/g tissue weight) 1 Liver Kidney Brin 8 6 d Triglyeride Conentrtions (mg/g tissue weight) 1 Liver Kidney 8 6 Brin Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 9 Effets of rsenite nd rsente on hepti, renl nd rin triglyeride onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5

10 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 1 of 15 8 Hert Lung Spleen 8 Hert Lung Spleen Triglyeride Conentrtions (mg/g tissue weight) 6 Triglyeride Conentrtions (mg/g tissue weight) 6 d Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 1 Effets of rsenite nd rsente on hert, lung nd spleen triglyeride onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5 At physiologil ph, trivlent rseni ompounds re neutrl in hrge while pentvlent rsenils re negtively hrged. Trivlent rseni speies n thus, redily trnsverse ell memrnes thn do pentvlent speies [6]. Arsenite, therefore, might hve een deposited in the orgns t fster rte thn rsente. Aprt from lood, spleen umulted the highest onentrtion of rseni thn ny other tissue studied in rsenite-exposed rts. This ws followed y hert nd then kidney, rin, lung nd liver in tht order. In rsente-exposed rts, umultion ws highest in the kidney, followed y lung, hert, rin, spleen nd liver. Arsente, in its protonted form, hs similr properties nd n sustitute for phosphte in severl iohemil retions [7]. The high deposition in the kidney ould, therefore, hve resulted from the orgn s physiologil involvement in the resorption of phosphte. Arsente nd phosphte hve een reported to ompete for the sme inding sites, with preferene for rsente sorption [8]. One tken up y the tissues, inorgni rseni is methylted to MMA V nd DMA V nd exreted in the urine []. However, this iomethyltion proess my eome sturted due to the different levels of methyltrnsferses present in eh tissue, resulting in redued rte of lerne nd hene inresed deposition in the tissues [9]. Furthermore, this iomethyltion proess (oxidtive methyltion followed y redution to trivleny) whih ws originlly onsidered detoxifition proess, my tully e n tivtion proess in view of the formtion of retive intermedites like MMA III nd DMA III [3]. In ddition to these, improvement in nlytil tehniques hs led to the disovery of new sulphur-ontining Phospholipid Conentrtions (mg/g tissue weight) 6 Liver Kidney Brin Phospholipid Conentrtions (mg/g tissue weight) 6 Liver Kidney Brin Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 11 Effets of rsenite nd rsente on hepti, renl nd rin phospholipid onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5

11 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 11 of 15 Hert Lung Spleen Hert Lung Spleen 5 5 Phospholipid Conentrtions (mg/g tissue weight) Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 1 Effets of rsenite nd rsente on hert, lung nd spleen phospholipid onentrtions. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5 Phospholipid Conentrtions (mg/g tissue weight) methylted rseni metolites, monomethylmonothiorsoni id (MMMTA V ) nd dimethylmonothiorsini id (DMMTA V ) in humn urine [31]. To wht extent this reent rseni toxiokineti informtion influenes rseni distriution/deposition in tissues on exposure to inorgni rseni through drinking wter remins to e eluidted. The findings of this study lso indite tht suhroni exposure to rsenite nd rsente through drinking wter is ssoited with lipotoxi nd nonlipotoxi perturtions in lipid homeostsis in orgns, lipoproteins, plsm nd erythroytes s well s inrese in HMG CoA redutse tivity. Compred to ontrol nimls, lipotoxi perturtions in rseni-exposed nimls were hrterised y high irulting FFA, enhned spleni holesterogenesis nd phospholipidosis, pulmonry nd hepti holesterogenesis s well s hypertriglyeridemi in plsm nd LDL + VLDL frtion. Derese in pulmonry nd hepti phospholipids, depletion of rdi nd rin lipids s well s depletion of erythroyte, plsm nd HDL holesterol nd phospholipids in irultion, hrterised the non-lipotoxi effets of oth rsenils. An enrihment with holesterol nd depletion of phospholipids were lso oserved in the LDL + VLDL frtion. Sine the ptterns of this 6 Liver Brin 6 Liver Brin HMG CoA/Mevlonte Rtio d HMG CoA/Mevlonte Rtio Control 5 ppm 1 ppm 15 ppm Arseni Dose (Sodium rsenite) Control 1 ppm 15 ppm ppm Arseni Dose (Sodium rsente) Fig. 13 Effets of rsenite nd rsente on hepti nd rin HMG CoA/ Mev-lonte rtios s n Index of HMG CoA redutse tivity. Eh r represents the men ± S.E.M. of 7 rts. Brs with different lphets re signifintly different t p <.5

12 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 1 of 15 Tle 3 Assoition etween tissue rseni levels nd some lipid prmeters in the nimls on exposure to sodium rsenite nd sodium rsente for 1 weeks through drinking wter Plsm FFA HDL holesterol Hepti HMG CoA redutse Brin HMG CoA redutse Prmeter Correltion oeffiient (r) Correltion oeffiient (r) Correltion oeffiient (r) Correltion oeffiient (r) Liver rseni NS.38 Kidney rseni Brin rseni NS.556 Blood rseni NS.58 Signifint t p <.5 NS-Not Signifint homeostti imlne differ with eh rseni speie nd dose, different mehnisms might medite this imlne. In ddition, sine exposure to the rseni speies ws through the orl route, intertion etween rseni nd these lipids proly did not egin t the level of the gstro-intestinl trt. Rther, lipid dynmis fter sorption might e the lipotoxi trget of rseni. These oservtions ould e disussed long severl lines. Under norml physiologil onditions, ll tissues vidly quire lipids from irulting non-esterified FFA ssoited with lumin, esterified ftty ids ound to lipoproteins hylomirons nd very low density lipoproteins (nd lierted y lipoprotein lipse-medited lipolysis), internliztion of whole lipoproteins nd de novo synthesis [3, 33], lthough de novo synthesis might ply only minor role in some tissues [3, 3]. Exess lipid, eyond tht needed for ellulr strutures nd ATP genertion is stored in lipid droplets [3, 3]. However, tissues like liver, intestine, hert nd rin re le to synthesise nd serete exess lipids in lipoproteins [3, 35 38]. FFAs originting from either lumin or lipoproteins enter the orgns either y pssive diffusion or vi protein rrier-medited pthwy [33, 3]. While the former is lower ffinity non-sturle proess operting t higher FFA onentrtions, the protein rrier-medited pthwy is low pity ut high ffinity proess operting t FFA/lumin rtios found in the plsm [33, 3]. Protein rriers tht hve een hrterized inlude ftty id trnslose (FAT/CD 36), plsm memrne ftty-id inding protein nd ftty id trnsport protein [33, 3]. Both rseni speies indued signifint elevtion of plsm FFA onentrtion. This oservtion implies tht while rseni exposure did not ffet the sorption of dietry lipids, ellulr lipid dynmis ws modulted y rseni exposure. Generlly hydrolysis of triglyerides ours in the dipose tissue nd results in the prodution of FFA with its susequent relese into the plsm. The elevtion of this lipid suggests its inresed moiliztion from the dipose tissue whih ould e indued y physiologil nd psyhologil stress [39]. With onsistent report of rseni using oxidtive stress [ ], the elevted plsm FFA implies n rseniindued ugmenttion of triglyeride hydrolysis in the dipose tissue y triglyeride lipse, resulting in inresed moiliztion of the lierted FFA into the plsm. The physiologil onsequenes of this elevted plsm FFA ould e viewed from the metoli roles of FFA. While this elevted plsm FFA should provide n immedite sustrte for triglyeride synthesis s well s the soure of ville fuel for the tissues nd lso the neessry signl for tissues to oxidize them [3, ], dt in Fig. 6 indite wht while exposure to rseni did not inhiit the uptke of FFA y the tissues, onsiderle mount of the FFA ws direted towrds the synthesis of triglyerides in the liver, kidney nd lungs s well s phospholipids in the spleen. Dt in Fig. 9 lso indite tht while the liver ould e sid to hve limited pity for triglyeride storge, tissues like kidney nd lungs umulted the triglyeride to out -fold tht of the liver. This further suggests tht rseni indued dysfuntion of triglyeride degrdtion seondry to insuffiient mitohondril ß-oxidtion of FFA, thus ompromising energy metolism in these tissues. Further signifint energeti nd funtionl onsequenes might e expeted. The key enzyme in the distriution of irulting lipids etween orgns is lipoprotein lipse (LPL), n enzyme loted on the wlls of lood pillries [3]. The role of LPL in lipoprotein metolism is well known. Sine mjority of the irulting FFAs re present s triglyerides in lipoproteins, hydrolysis of this triglyeride y LPL is n importnt determinnt of overll ftty id uptke nd ß-oxidtion in the tissues [5, 6]. High irulting FFA (s oserved in this study) is known to inhiit the tivity of LPL [7]. Homeosttilly, inhiition of LPL is expeted to hve the following onsequenes on lipid dynmis in the irultion: 1. Severe hypertriglyeridemi seondry to underutilistion of hylomirons nd VLDL. Hypoholesterolemi 3. Derese in HDL holesterol (otherwise known s reverse holesterol trnsport) nd phospholipids [8].

13 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 13 of 15 Tht rseni exposure resulted in ll these lipid metoli derngements (Figs. 1 nd 3) suggests tht the inhiition of LPL might medite these oserved effets of rseni. Another mjor finding of this study ws the depletion of rdi lipids in rseni-exposed nimls. Physiologilly, rdi myoytes hve regultory pthwys tht regulte lipid metolism. The myordium hs lile stores of triglyeride tht serve s n endogenous soure of FFAs [9, 5]. Intrmyordil triglyeride n e hydrolysed y hormone sensitive lipse nd dipose triglyeride lipse [3, 33]. Insulin inhiits lipolysis, wheres teholmines, thyroid hormone nd glugon, elerte intrmyordil triglyeride degrdtion [51]. Whether rseni-indued drenergi stress in the nimls might medite the depletion of rdi lipids oserved in this study is not known t present. Furthermore, sine the mjor rdi lipids were depleted s result of rseni exposure, the possiility tht rseni might indue overexpression/prodution of rdi polipoprotein B (Apo B) whih inresed lipid seretion from the hert, nnot e ruled out [38]. Apo B expression hs een found to inrese in herts of ptients with oronry rtery disese [5], rdiovsulr outome tht is hllmrk of rseni exposure [53]. Due to the lood rin rrier, irulting lipoproteins nnot reh the rin exept for smll HDL prtiles [5]. Most of the lipoproteins in the rin re synthesised y the stroytes nd hve een postulted to e responsile for the trnsfer of lipids within the rin [36, 55]. Thus, n rseni-indued dmge to the lood rin rrier might e responsile for the dyslipidemi oserved in the rin of the rseni-exposed nimls [56, 57]. Enhned holesterogenesis oserved in the spleen, lungs nd liver of rsenite- nd rsente-exposed nimls s well s the kidney of rsente-exposed nimls, my e ttriuted to n rseni-indued tivtion of 3-hydroxy-3-methylglutryl Coenzyme A (HMG CoA) redutse (the rte limiting enzyme in holesterol synthesis) or it my e due to feedk inhiition [58, 59]. It my lso e due to inhiition of the tivity of holesterol-7α-hydroxylse, ytohrome P5 enzyme loted in the endoplsmi retiulum. This ould limit the iosynthesis of ile ids, whih is the only signifint route for elimintion of holesterol from the ody [6]. Sine the liver hs limited pity to store lipids, the exess holesterol nd triglyerides re pkged into VLDL prtiles nd sereted into irultion. Consistent with this ws the oservtion of the enrihment of LDL + VLDL frtion with holesterol nd triglyerides. Furthermore, sine this frtion ws depleted of phospholipids, this implies filure osioned y rseni exposure in hepti provision of phospholipids tht re found in lipoproteins (Fig. ) [61]. Compelling experimentl dt from niml studies hve shown positive ssoition etween high irulting FFA nd pthologil onditions suh s dietes mellitus, oesity, thyrotoxiosis, theroslerosis, oronry rtery disese, rdi rrhythmi, high lood pressure nd pulmonry diseses [39, 6 6]. Furthermore, elevted pulmonry holesterol hs een shown to inhiit surftnt funtion y disrupting the physiology nd turnover of surftnt, leding to impirment of lungs mehnis [65]. Anormlities in surftnt metolism re the leding uses of ute respirtory distress syndrome, ute lung injury nd diverse rry of other respirtory illnesses [66]. Tht rseni exposure uses ll these onditions hve een onfirmed from epidemiologil studies of rseniosis [3]. Thus, the sustined dyslipidemi oserved in this study my e one of the underlying mehnisms of these rseni-indued pthologies. One limittion of this study ws tht rseni speition ws not rried out in the tissues. Rther, rseni in the tissues ws determined s totl rseni. In view of the reent disovery of new methylsulphur derivtives of inorgni rseni, it opens up the possiility tht these orgni metolites my e more sutle nd insidious in their toxi effets. Toxiologil informtion on these newly disovered orgni metolites is snty. However, in reent study in our lortory in whih rts were exposed to pentvlent inorgni nd orgni rsenils through drinking wter, rseni umultion nd dyslipidemi of the sme mgnitude were lso oserved. These reent dt, together with the dt in the present study, support linkge etween metolism nd hemil form of rseni nd lipotoxi nd non-lipotoxi effets of this metlloid. Conlusion In onlusion, even though su-hroni exposure to rsenite nd rsente through drinking wter produed different ptterns of dyslipidemi, our study identified two ommon denomintors of dyslipidemi nmely: inhiition of reverse holesterol trnsport nd inrese in plsm FFA. These two denomintors (in ddition to other individul perturtions of lipid metolism indued y eh rsenil), might medite the oserved rdiovsulr nd other disese endpoints of inorgni rseni exposure through drinking wter. Arevitions HDL: High density lipoproteins; FFAs: Free ftty ids; HMG CoA redutse: 3-hydroxy-3-methylglutryl oenzyme A redutse; LDL: Low density lipoproteins; VLDL: Very low density lipoproteins; MMA: Monomethylrsoni id; DMA: Dimethylrsini id; MMMTA: Monomethylmonothiorsoni id; DMMTA: Dimethylmonothiorsini id; FAT: Ftty id trnslose; LPL: Lipoprotein lipse. Competing interests The uthors delre tht they hve no ompeting interests

14 Afoli et l. BMC Phrmology nd Toxiology (15) 16:15 Pge 1 of 15 Authors ontriutions OA, EAB nd OOOD (Odukoy) oneived the work. OA designed the work. OA, EAB nd OOOD oordinted the study. OKA, ADW, OOOG (Ogunrinol), EOA, DOB, OAD, OBO nd OA were involved in lipid nd metl nlyses. OKA nd OA drfted the mnusript nd did sttistil nlysis. All uthors red nd pproved the mnusript for sumission. Aknowledgements The tehnil ssistne of Mr. OA. Awoyemi, Mr. OJ. Olurinde, Mrs. JO. Adew nd Mrs. T. Osinjo is gretly knowledged. Author detils 1 Deprtment of Biohemistry, Federl University of Agriulture, Aeokut, Nigeri. Deprtment of Chemistry, Federl University of Agriulture, Aeokut, Nigeri. 3 Deprtment of Biohemistry, Ldoke Akintol University of Tehnology, Ogomosho, Nigeri. Deprtment of Biohemistry, Lgos Stte University, Ojoo, Lgos, Nigeri. 5 Deprtment of Biohemistry, University of Ilorin, Ilorin, Nigeri. 6 Biohemistry Unit, Deprtment of Chemil Sienes, Bells University of Tehnology, Ot, Nigeri. 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