COMPARATIVE PROTECTIVE POTENTIALS OF COMPARATIVE PROTECTIVE POTENTIALS OF VITAMINS C AND E AGAINST NITROCELLULOSE VITAMINS C AND E AGAINST NITROCELLULOSE

THINNER-INDUCED ATHEROGENICITY IN MALE AND THINNER-INDUCED ATHEROGENICITY IN MALE AND FEMALE ALBINO FEMALE ALBINO WISTARWISTAR RATS. RATS.

PRESENTED BYPRESENTED BY

FRIDAY E. UBOH,FRIDAY E. UBOH, PhD, MIPAN PhD, MIPANASSOCIATE PROFESSOR OF BIOCHEMISTRYASSOCIATE PROFESSOR OF BIOCHEMISTRY

(TOXICOLOGY) (TOXICOLOGY)

BIOCHEMISTRY DEPARTMENTBIOCHEMISTRY DEPARTMENTFACULTY OF BASIC MEDICAL SCIENCESFACULTY OF BASIC MEDICAL SCIENCES

UNIVERSITY OF CALABARUNIVERSITY OF CALABARCALABAR - NIGERIACALABAR - NIGERIA

E-mail: fridayuboh@unical.edu.ng & E-mail: fridayuboh@unical.edu.ng & fridayuboh@yahoo.comfridayuboh@yahoo.com

INTRODUCTIONINTRODUCTIONThere is an increasing concern about

voluntary and involuntary exposure to solvents that generate pollutants into the environments.

Exposure to these pollutants can occur from intentional or unintentional ingestion or inhalation of such solvents.

Both acute intentional and chronic unintentional exposure to industrial solvents for may cause serious intoxicating and health problems.

Nitrocellulose thinner (NCT) is one of such solvents of abuse among delinquent youths in some societies (Ho et al., 1998; Dodd, 2001; MacLean and D’Abbs, 2002; Anderson and Loomis, 2003; Wille and Lambert, 2004).

NCT, in the form of lacquer thinner, is an industrial solvent commonly used in furniture, paint and automobile spray painting industries (Patrick-Iwuanyawu et al., 2013).

• The solvent contains different organic chemical substances, such as ethylbenzene or toluene and butyl acetate.

• These chemical substances are known to constitute chemical pollutants in different environments.

• WHO (2005) reported that these chemical pollutants are detectable in household and workplace air.

• Hence, exposure to chemical pollutants from NCT in indoor and outdoor environments may be common.

•

• Possible mode of exposure include; (i) Oral ingestion of drinks and foods contaminated by the solvent in course its usage; (ii) Inhalation of the contaminated air at and around the occupational environments; (iii) Dermal exposure from spillage during usage.

• Manufacturers, furniture and automobile spray painters, as well as those residing around these work environments, therefore tend to be the population with greater risk of exposure.

Introduction Cont’d

Particularly, occupational exposure to mixtures of toluene, ethylbenzene and butylacetate have been reported in painting or lacquering workplaces (Seeber et al., 1996; Jovanovic et al., 2004; Faber et al., 2006).

Different health and environmental hazards have been shown to be associated with exposure to some industrial solvents, including NCT (Abousalem et al., 2012; Uboh et al., 2012a-c, 2013, 2014a,b; USEPA, 1998; Burns et al., 1994).

Introduction Cont’d

Introduction Introduction Cont’dCont’dParticularly, exposure to NCT and related

organic solvents has been reported to induce haematotoxicity, hepatotoxicity and nephrotoxicity in humans and experimental animals (Jovanovic et al., 2004; Faber et al., 2006; Patil et al., 2007; Uboh et al., 2012a,b; 2013; 2014a,b).

The likely cause of NCT toxicity has been suggested to be the adverse interaction between the constituents of the solvent or their reactive metabolites and various macromolecules in the body tissues (Patrick-Iwuanyawu et al., 2013; Uboh et al., 2012a,b; Dioka et al., 2004; Wang et al., 2002).

Measures of ameliorating or protecting the body against chemical induced toxicity have an issue of major concern in the recent times.

Information on the possible protective agents against NCT induced toxicities is inadequate.

However, protective effect of vitamins E and C against gasoline and NCT induced hepato- and nephrotoxicities in rats have been earlier reported (Uboh et al., 2012c, 2014c).

Introduction Cont’d

Introduction Introduction Cont’dCont’d

This study therefore assessed the comparative protective potentials of vitamins C and E against NCT-induced atherogenicity in male and female albino Wistar rats.

MATERIALS AND METHODS:MATERIALS AND METHODS:Animal Handling and Treatment: Forty eight matured male and female

albino Wistar rats (24 males and 24 females), weighing180 to 200g were used in the study.

Rats were obtained from Biochemistry Department Experimental Research Animal House of the University of Calabar, Calabar, Nigeria.

The animals were allowed free access to standard laboratory diet and tap water, ad libitum.

The work was carried out under 12 hours light/dark cycle illumination and prevailing tropical room temperature.

Preliminary acute toxicity studies in mice, gave LD50 of 160.2mg/kg body weight of nitrocellulose thinner solvent.

Hence, 48.0mg/kg body weight (30% of LD50) were orally administered to rats in test groups for 30 days as previously described (Uboh et al., 2012a,b; 2013; 2014a,b).

Materials and Methods Cont’d

Materials and Methods Materials and Methods Cont’dCont’d

The animals were distributed into eight groups, with six rats each, as highlighted below:

Group 1a: Negative Control I, comprised of six male rats receiving 1.0ml/kg body weight of distilled water placebo for 30 days

Group 1b: Negative Control II, comprised of six female rats receiving 1.0ml/kg body weight of distilled water placebo for 30 days

Group 2a: Positive Control I, comprised of six male rats receiving 48.0mg/kg body weight of nitrocellulose thinner (NCT) for 30 days.

Group 2b: Positive Control II, comprised of six female rats receiving 48.0mg/kg body weight of nitrocellulose thinner (NCT) for 30 days.

Materials and Methods Materials and Methods cont’dcont’d

Group 3a: Comprised of six male rats receiving 48.0mg/kg body weight of NCT + 200mg/kg body weight of vitamin C for 30 days

Group 3b: Comprised of six female rats receiving 48.0mg/kg body weight of NCT + 200mg/kg body weight of vitamin C for 30 days

Group 4a: Comprised of six male rats receiving 48.0mg/kg body weight of NCT + 200IU/kg body weight of vitamin E for 30 days

Group 4a: Comprised of six female rats receiving 48.0mg/kg body weight of NCT + 200IU/kg body weight of vitamin E for 30 days

The animals were sacrificed, 24 hours after the 30th day of experimental period.

All animal experiments were carried out according to the Guidelines of Institution’s (University of Calabar, Nigeria) Animal Research Ethics Committee, with reference to the Guide for the Care and Use of Laboratory Animals (NRC, 1995).

Materials & Methods Cont’d

Collection and preparation of blood for analyses:

Blood samples were collected from the sacrificed by cardiac puncture, under chloroform vapor anaesthesia into heparinised sample bottles.

The blood samples were centrifuged with Table-top centrifuge (MSE model, England) at 3000 rpm for 5 minutes to obtain the plasma, which was used for the biochemical assays.

Materials & Methods Cont’d

•Biochemical analyses: The plasma lipid profile. including total cholesterol (TC), triacylglycerol (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) were determined using referenced standard methods.

• Plasma lipid ratios, including TC:HDL, TG:HDL, (TC–HDL)/HDL and Log(TC/HDL) were also calculated to determine the atherogenic level.

Materials & Methods Cont’d

Materials & Methods Materials & Methods Cont’dCont’d

• Reagent kits obtained from Biosystems Laboratories (S. A. Costa Brava, Barcelonia, Spain) were used in the study.

•And all the reagent kits were of analytical grade.

Statistical Analysis: Results were presented as mean ± S.E.M.

The data generated from the study were statistically analysed using one-way analysis of variance (ANOVA) with SPSS window statistical software programme.

Student "t" test was also used for pair-wise comparison, and differences were considered significant at p<0.05.

Materials & Methods Cont’d

RESULTS:RESULTS: The results of this study are presented

in Figures 1 - 4, and Table 1.

The results showed that the levels of plasma TC, TG, LDL, and the atherogenic indices, (TC–HDL)/HDL and Log(TC/HDL), recorded for male and female rats exposed to NCT (positive control groups) were significantly (p<0.05) higher, while HDL levels were significantly (p<0.05) lower, compared respectively to the levels recorded for rats in the respective negative control groups.

Results Results cont’dcont’d

The recorded NCT-induced percentage increase in TC, TG, LDL, and decrease in HDL for female rats (96.35±4.86, 96.75±5.21, 96.36±5.66 and 85.91±6.27 percents respectively) were however insignificantly (p>0.05) higher, compared to the percentages recorded for males (89.91±5.82, 89.86±6.73, 89.86±6.88 and 80.93±9.34 percents respectively).

The increase in TC, TG, (TC–HDL)/HDL and Log(TC/HDL) and decrease in HDL levels reported for rats exposed to NCT were therefore observed to be sex-independent.

Results Results cont’dcont’d

• The results also showed that concomitant treatment of the respective groups of rats exposed to NCT with vitamins C and E significantly (p<0.05) protected both the male and female rats against NCT-induced atherogenicity.

• However, the protection potential of vitamin E against NCT-induced atherogenicity was observed to be significantly (p<0.05) higher than that of vitamin C in both sexes of rats.

Table 1. Table 1. Comparative effect of Vitamins C and E on some Comparative effect of Vitamins C and E on some plasma lipid ratios (atherogenic indices) of male and female plasma lipid ratios (atherogenic indices) of male and female rats orally exposed to NCT.rats orally exposed to NCT.Group

Treatment

TC:HDL TG:HDL (TC–HDL)/HDL Log(TC/HDL)

1a (M)

Water 1.17±0.04

1.09±0.05

0.17±0.03 0.07±0.01

1b (F) Water 1.21±0.04

1.05±0.06

0.21±0.03 0.08±0.01

2a (M)

NCT only 4.03±0.88*

3.74±0.92*

3.03±0.14* 0.61±0.02*

2b (F) NCT only 4.40±1.06*

3.85±0.11*

3.85±0.11* 0.64±0.02*

3a (M)

NCT + Vit C

2.07±0.09

1.92±0.06

1.07±0.04 0.32±0.03

3b (F) NCT + Vit C

2.20±0.68

2.11±0.10

1.20±0.04 0.34±0.02

4a (M)

NCT + Vit E

1.52±0.03a

1.41±0.04a

0.52±0.06a 0.18±0.01a

4b (F) NCT + Vit E

1.62±0.03a

1.55±0.07a

0.62±0.07a 0.21±0.01a

Values are presented as mean ± SEM; n = 6; M = Males, F = Females; *P<0.05 compared respectively to 1a, 1b, 3a, 3b, 4a & 4b; ap<0.05 compared to 3a & 3b.

CONCLUSIONCONCLUSION:: The results of this study suggested that oral exposure to NCT introduced some chemical agents into the body tissues.

And that these agents, or their metabolites, interacted with the body tissues to induce atherogenicity in a sex-independent pattern.

The results also indicated that vitamins C and E may provide protection against NCT-induced atherogenicity in male and female rats.

And that vitamin E is relatively more potent than vitamin C in providing protection against NCT-induced atherogenicity in both male and female rats.The results of this study correlated with the results of our earlier studies on the hepatoprotective effect of vitamins C and E against gasoline vapor-induced liver injury and NCT-induced nephrotoxicity in male rats (Uboh et al., 2012c; 2014c).

Conclusion Cont’d

Based on the results of this study, it may be concluded that vitamins C and E are capable of providing protection against NCT-induced atherogenicity in male and female rat model;

And that vitamin E showed a relatively higher potency than vitamin C, in this regard.

Conclusion Cont’d

THANK THANK YOU YOU

ACKNOWLEDGEMENTACKNOWLEDGEMENTAll my Post graduate students.All the technical staff of

Biochemistry Department, University of Calabar, Nigeria.

REFERENCES:REFERENCES:Abousalem, M., Elgerwi, A. and El-Mashad, A. (2012).

Biotoxic and haemotoxic effects of air pollutants at a Benzene station on albino rats. Int. J. Anim. Veter. Adv., 4(3): 187-190.

Anderson, C. and Loomis, G. (2003). Recognition and prevention of inhalant abuse. American Family Physician. 68(5): 869-874.

Dodd, J. (2001). Petrol sniffing in a pregnant Aboriginal population: A review of maternal and neonatal outcomes. The Australian and New Zealand Journal of Obstetrics and Gynaecology. 41(4): 420-423.

Faber, W. D., Roberts, L. S. G., Stump, D. G., Tardif, R., Krishnan, K., et al. (2006) Twogeneration reproduction study of ethylbenzene by inhalation in Crl-CD rats. Birth Defects Res B Dev Reprod Toxicol 77: 10-21.

Ho, W., To, E., Chan, E., & King, W. (1998). Burn injuries during paint thinner sniffing. Burns. 24(8): 757-759.

Jovanović, J. M., Jovanović, M. M., Spasić, M. J. and Lukić, S. R. (2004) Peripheral nerve conduction study in workers exposed to a mixture of organic solvents in paint and lacquer industry. Croat Med J 45: 769-774.

MacLean, S. & D’Abbs, P. (2002). Petrol sniffing in Aboriginal communities: A review of interventions. Drug and Alcohol Review. 21(1): 65-72.

Patil, A. J., Bhagwat, V. R., Patil, J. A., Dongre, N. N., Ambekar, J. G. and Das, K. K. (2007). Occupational lead exposure in battery manufacturing workers, silver jewelry workers, and spray painters in Western Maharashtra (India): effect on liver and kidney function. J. Basic Clin. Physiol. Pharmacol. 18(2): 87-100.

Patrick-Iwuanyanwu, K. C., Okon, E. A., Areh, N. W. and Wegwu, M. O. (2013). Toxicological effect of inhalation exposure to nitrocellulose paint thinner fumes (FIAB®, ABRO® and SPRINT®) in wistar albino rats. Arch. Appl. Sci. Res. 5 (1):264-269 .

References Cont’d

Seeber, A., Sietmann, B. and Zupanic, M. (1996) In search of dose-response relationships of solvent mixtures to neurobehavioural effects in paint manufacturing and painters. Food Chem Toxicol 34: 1113-1120.

Uboh, F. E., Usoh, I. F., Nwankpa, P. and Obochi, G. O. (2012a). Effect of Oral Exposure to Nitrocellulose Thinner on Haematological Profiles of Male Albino Wistar Rats. American Journal of Biochemistry and Molecular Biology 2(4): 227-234

Uboh, F. E., Akpanabiatu, M. I., Aquaisua, A. N. and Bassey, E. I. (2012b). Oral Exposure to Nitrocellulose Thinner Solvent Induces Nephrotoxicity in Male Albino Wistar Rats. Journal of Pharmacology and Toxicology 7(2): 78-86

Uboh, F. E., Ebong, P. E. , Akpan, H. D. and Usoh, I. F. (2012c). Hepatoprotective effect of vitamins C and E against gasoline vapor-induced liver injury in male rats. Turkish Journal Biology, 36: 217-223.

References Cont’d

Uboh, F. E. and Ufot, S. (2013) Withdrawal from exposure reverses hematotoxicity and hepatotoxicity caused by oral exposure to Nitrocellulose thinner in male rats. Journal of Clinical Toxicology 3: 173. doi:10.4172/2161-0495.1000173.

Uboh, F. E., Ufot, S., Mboso, S. and Eyong, E. U. (2014a). Effect of Costus afer Leaves’ Juice on Nitrocellulose Thinner Induced Nephrotoxicity in Rats. Research Journal of Environmental Toxicology 8 (1): 37-45.

Uboh, F. E., Mboso, E., Ufot, S., Igile, G. O. and Ebong P. E. (2014b). Hepatotoxicity effect of oral exposure to Nitrocellulose thinner in albino Wistar rats. The Journal of Toxicology and Health. Photon 104, 448-455.

Uboh, F. E. (2014c). Effect of combined administration of vitamins C and E on some renal functions indices of rats exposed to nitrocellulose thinner. OMICS International Conference on Toxicology, Chicago.

References Cont’d

Wille, S. and Lambert, W. (2004) Volatile substance abuse: Post mortem diagnosis. Forensic Science International. 142(2-3): 135-156

World Health Organization (WHO). (2005). Concise International Chemical Assessment Document 64. Butyl Acetates. World Health Organization: Geneva.

References Cont’d