the consequences of in utero exposure to lead acetate on exposure ...

LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLII (2), 2009, TIMIŞOARA

THE CONSEQUENCES OF IN UTERO EXPOSURE TO LEAD ACETATE ON EXPOSURE AND INTEGRITY BIOMARKERS OF REPRODUCTIVE SYSTEM IN FEMALE RATS AT SEXUAL MATURITY DUMITRESCU EUGENIA, TRIF ALEXANDRA, ARGHERIE DIANA, R.T. CRISTINA Faculty of Veterinary Medicine Timisoara, 119Calea Aradului, 300645, Timisoara, Romania E-mail: [email protected] Summary

The aim of the study was the evaluation of lead toxic impact on the female reproductive system integrity and exposure biomarkers, because of lack of researches and contests related to the opinions regarding lead toxicity on the reproduction function in females and presences in Romania of the pollutant lead industry. The objectives of this research were the evaluation of lead levels in ovary and uterus (exposure biomarker) and structural changes of the genital apparatus (ovary, uterus) (integrity biomarkers). From the results of the studies results that exposure to lead acetate in utero period of female rats determined in adult period. Key words: lead, rats, ovary, uterus, cervix, histoarhitectonic.

The exposure to lead acetate in utero period of female rats determined in adult period: significant increase of lead level in ovaries, Fallopian tubes and uterus compared with the control group and in direct correlation, with different degrees of significance, with the exposure level; severe congestive and degenerative changes in ovary and uterus. The studies concerning reproductive toxicity are more and more in researcher’s attention. The consequences of the exposure to industrial contaminants – lead, especially in females are limited and, sometimes controversially (1, 3, 5). Materials and methods The study was carried out on 28 adult female rats (90 days) exposed to lead during gestation/in utero, period as follows: E1: 50 ppb Pb (the maximum admitted level in drinking water according to the Law 485/2002); E2: 100 ppb Pb; E3: 150 ppb Pb. In the case of offspring, the exposure to lead was stopped from birth until sexual maturity. Control group received tape water. The forages and water have been assured ad libitum. All assays with animals were conduced in accordance with present law regarding animal welfare and ethics in animal experiments 295


(143/400/2002; 471/2002; 206/2004; 9/2008; 86/609/CEE). At sexual maturity the female offspring were sacrificed following protocols and ethical procedures and ovary, Fallopian tubes and uterus were taken of for lead level determination and histological exam. The lead level was determined in genital organs (ovary, Fallopian tubes and uterus) by of atomic absorption spectrophotometry in the Laboratory of Nutrition and Toxicology from Faculty of Veterinary Medicine Timisoara, with the spectrometer AAS AA-6650 Shimadzu, with graphite oven, provided by the company Viola Bucharest and the structural changes on histological section trichromic Mallory stained (after fixation in alcohol 80°, sectioning at 5µ). The results were statistically processed by the software Anova and the Student test. Results and discussions The results regarding lead level in genital organs (ovary, uterus and Fallopian tubes) are summarized in table 1 and fig.1. The study emphasized: higher, significant accumulation in genital organs (E groups) comparative to C group and in direct correlation with exposure level (ovary: E1/C: +169.9%; E2/C: +278.78%; E3/C: +363.63%; E2/E1: +40.44%; E3/E2: +22.4%; E3/E1:+71.91%; uterus: E1/C: +125.68%; E2/C: +171.55%; E3/C: +180.73%; E2/E1: +20.32%, p>0.05; E3/E2: +3.37%, p>0.05; E3/E1:+24.39, p>0.05%). Table 1 Lead acetate average concentration (µg/g) in ovary, uterus and Fallopian tubes

Uterus and Fallopian tubes

Ovary

Organs

Group C E1 E2 E3 XE C E1 E2 E3 XE

X±Sx 1.32±0.03 3.56±0.15 5.00±0.21 6.12±0.29 4.89 1.09±0.06 2.46±0.19 2.96±0.20 3.06±0.07 2.82

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DS 0.08 0.41 0.55 0.76 0.16 0.49 0.53 0.19 -

Confidence level 95% 0.40 0.40 0.40 0.40 0.30 0.30 0.30 0.30 -

LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLII (2), 2009, TIMIŞOARA C

7

E1

6

E2

5

E3

4 3 2 1 0 Ovary

Uterus and Fallopian tubes

Fig.1. Dynamics of lead acetate levels Exposure to lead acetate determined severe structural changes in genital organs: ovary: follicular cells dispersion, vacuolar epithelial cells, destruction of zone parenchymatosa; uterus: necrosis of uterine glands, destruction of uterine lining cells. In present study were not observed ovarian cystes, changes emphasized by Hildebrad et al., (1989) quoted by National Research Council (3) and not many atrezic follicles mentioned by (2, 5) (2). The histological images are presented in the figure 2-7.

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B

Fig 2. Histological section in rats ovary after exposure to 50 ppb Pb, trichromic Mallory stain, X 100 Follicular cells dispersion (A); vacuolar epithelial cells (B) 297


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B

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Fig 3. Histological section in rats uterus after exposure to 50 ppb Pb F1 , trichromic Mallory ,stain X 200 Necrosis of uterine glands (A); destruction of uterine lining cells (B)

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Fig 4. Histological section in rats ovary after exposure to 100 ppb Pb, trichromic Mallory stain, X 100 Follicular cells dispersion (A); vacuolar epithelial cells (B) 298


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Fig 5. Histological section in rats uterus after exposure to 100 ppb Pb, trichromic Mallory stain, X 200 Necrosis of uterine glands (A)

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Fig 6. Histological section in rats ovary after exposure to 150 ppb Pb, trichromic Mallory stain, X 100 Destruction of zona parenchymatosa (A), vacuolar epithelial cells (B) 299


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B A

A Fig 7. Histological section in rats uterus after exposure to 100ppb Pb F1 Pb trichromic Mallory stain, X 200 necrosis of uterine glands (A); destruction of uterine lining cells (B) Conclusions Exposure to lead acetate in utero period of female rats determined in adult period: • Significant increase of lead level in ovaries, Fallopian tubes and uterus compared with the control group and in direct correlation, with different degrees of significance, with the exposure level; • Severe congestive and degenerative changes in ovary and uterus. References 1. Andrews, J.S., Biologic Monitoring and Biomarkers, ATSDR-Hazardous Waste Conference, 1993, http://www.atsdr.cdc.gov/cx6a.html. 2. Laxmipriya, P., Nampoothiri., Sarita, Gupta, Simultaneous of effect of lead and cadmiu on granuloasa cells: a cellular model for ovarian toxicity, Reprod. Toxicol, 2006, 21, 2, 179-185. 3. National Research Council, Biologic Markers in Reproductive Toxicology, National Academy Press, Washingthon, D.C., 1989. 4. US EPA, Lead Compounds, 2002, http://www.epa.gov/ttn/atw/hlthef/lead.html. 5. Taupeau, Crystel., Poupon., J., Nome Francoise., Brigitte Lefevre, Lead accumulation in the mouse ovary after treatment-induced follicular atresia. Reproductive Toxicology, 2001, 15, 4, 385-391. 300