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Original Article

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The effects of alpha-lipoic acid on breast of female albino rats exposed to malathion: Histopathological and immunohistochemical study

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Ola M. Omran a,b,∗,1 , Osama H. Omer c

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Department of Pathology, College of Medicine, Qassim University, Saudi Arabia Department of Pathology, College of Medicine, Assiut University, Egypt Department of Pharmaceutics, College of Pharmacy, Qassim University, Saudi Arabia

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a b s t r a c t

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Article history: Received 25 July 2014 Received in revised form 15 February 2015 Accepted 23 February 2015

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Keywords: Malathion ␣-Lipoic acid Rats Breast carcinoma

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Introduction

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Background: The wide use of the organophosphate insecticide malathion is accompanied by the risk of human exposure and may be involved in the etiology of breast cancers, especially in developing countries. Alpha (␣)-lipoic acid, a natural molecule, present in our diet has antioxidant and protective effects in cases such as aging, diabetes mellitus, and vascular and neurodegenerative diseases all in which free radicals are involved. However, there is only scarce data regarding the efficacy and biological activity of ␣-lipoic acid on malathion-induced breast histopathological changes. Aims: To investigate whether malathion can induce mammary histopathological changes, to immunohistochemically analyze the modulations in proliferation-apoptosis balance associated with these changes, to assess the associated metabolic parameters, antioxidant stress and hormonal profile changes and to elucidate the possible protective effect of ␣-lipoic acid on malathion induced alterations in rats. Materials and methods: Forty Wistar female rats weighing 150–170 g were divided into four groups. Group 1: control group were injected intraperitoneally (ip) with saline solution. Group2: animals were injected (ip) with malathion twice a day for five days. Group 3: animals were orally given ␣-lipoic acid, after three hours of treatment with malathion at the same dose given to group 2. Group 4: animals were treated with ␣-lipoic acid at the same dose given to group 3. Rats were sacrificed on the 90th day, and breast tissues were analyzed for histopathological and immunohistochemical alterations. Blood samples were collected for biochemical tests. Results: ␣-Lipoic acid exhibited a striking reduction of malathion-induced mammary tumor incidence, and reversed intra-tumor histopathological alterations. Alpha lipoic acid suppressed proliferating cell nuclear antigen (PCNA) and p53 expression, induced apoptosis, upregulated proapoptotic protein Bax. Conclusions: Our results provide the experimental evidence that ␣-lipoic acid exerts chemopreventive effect in the breast hyperplastic and malignant changes by suppressing abnormal cell proliferation and inducing apoptosis with an oncostatic effects during an early-stage breast cancer. © 2015 Published by Elsevier GmbH.

Breast cancer is considered major and common health problem in both developing and developed countries. The insecticide malathion which is widely used in agriculture and public health can generate certain harmful effects on humans. Hence, there is need

∗ Corresponding author at: Department of Pathology, Faculty of Medicine, Qassim University, Saudi Arabia. Tel.: +966 530664590. E-mail address: [email protected] (O.M. Omran). 1 Previous address: 4166 Pathology Department, The Ohio State University, Columbus, OH, USA.

for more research to be focused in this area. The etiology of breast cancers is dictated by both internal factors (inherited mutations, hormones, and immune conditions) and environmental/acquired factors (such as smoking, diet, drugs, radiation, and infectious organisms) [1,2]. Behavioral and environmental factors are considered to be among the major influencing components causing increase in the incidence of breast cancer risk [3]. In vivo and in vitro studies have shown that environmental substances (e.g., DDT, polychlorinated biphenyls, 4-nonylphenol, 4-octylphenol) can promote mammary cancer [4,5]. Organophosphorus compounds are organic ester of phosphoric or thiophosphoric acid and are increasingly used in agriculture, medicine, and industry. Because of growing concerns about health

http://dx.doi.org/10.1016/j.prp.2015.02.006 0344-0338/© 2015 Published by Elsevier GmbH.

Please cite this article in press as: O.M. Omran, O.H. Omer, The effects of alpha-lipoic acid on breast of female albino rats exposed to malathion: Histopathological and immunohistochemical study, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.02.006

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and environmental problems associated with pesticides, several governments are seeking to employ safe, convenient and economically feasible biological methods for combating pests. The organophosphorus compound malathion (O,O-dimethyl-S1,2-bis ethoxy carbonyl ethyl phosphorodithionate), is extensively used throughout the world to control a variety of outdoor insects in both agricultural and residential settings [6]. Malathion insecticide is widely used in Saudi Arabia to control motile stages of mites and some other insects on fruits and vegetables and has limited plant systemic activity [7,8]. Malathion is converted into malaoxon that inhibits acetylcholinesterase (AChE) [9]. Malathion has been shown to be mutagenic and there is suggestive evidence of carcinogenicity for malathion in animals [10]. The association between cancer risks in humans and agricultural pesticides has been studied in both occupational and nonoccupational exposures [11]. In vitro studies showed that malathion induced malignant transformation of breast epithelial cell lines [12]. A previous study showed synergistic effect of malathion and estrogen on mammary gland carcinogenesis [4,13]. Currently used pesticides such as malathion affect the function of sex hormone receptors thus possessing endocrinedisrupting potential [14]. There is a consensus that malathion induces toxicity and carcinogenicity through the inhibition of acetylcholinesterase (AchE) [2,12]. Moreover, several organophosphorus pesticides including malathion have been reported to cause oxidative stress [15]. Oxidative stress as defined by Dare et al. is an imbalance between the systemic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates [16]. Oxidative stress with increased production of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses is associated with production of reactive oxygen species (ROS). The reactive oxygen species released by the mitochondrial respiratory chain can damage a wide range of essential biomolecules resulting in lipid peroxidation, DNA damage and enzyme inactivation [17,18]. Antioxidant enzymes, mainly Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) are the first line of defense against free radical induced oxidative stress. SOD is known to convert superoxide radicals to H2 O2 and molecular oxygen [19]. Catalase as an important regulator of oxidative stress, is responsible for the catalytic decomposition of hydrogen peroxide to molecular oxygen and water [20,21]. GPx, responsible for enzymatic defense against hydrogen peroxide, it catalyses the reaction between glutathione and hydrogen peroxide to form glutathione disulphide (GSSG) and the reduction product of H2 O2 [22]. Resistance to chemotherapy is a major obstacle to successful treatment of breast cancer [23,24]. Earlier and current literature shows that over 60% of breast cancer patients use some form of complementary and alternative medicine [25,26]. Studies have shown that phytochemicals that display potent anticancer effects in both in vitro and in vivo rodent models can be potential candidates for chemoprevention in humans [27,28]. These phytochemicals have different chemical properties and can block tumorigenesis by multiple mechanisms that include prevention of pro-carcinogen activation, inhibition of cell proliferation, invasion, angiogenesis, and stimulation of apoptosis [29]. Alpha (␣)-lipoic acid (thioctic acid) is a naturally occurring dithiol compound. It was discovered in 1951. Alpha-lipoic acid catalyzes oxidative decarboxylation process converting pyruvate to acetyl CoA. Hence, ␣-lipoic acid is essential for energy production in cells. Lipoic acid is found mainly in animal foods such as meat and liver and at low or undetectable levels in plant foods such as potato [30,31]. There is some evidence for the protective effect of Alpha lipoic acid in cases such as rheumatoid arthritis, lyme disease, diabetes mellitus, and vascular and neurodegenerative diseases and age-related conditions in which free radicals are involved [32–36].

Alpha lipoic acid has also been suggested for cataracts, glaucoma, multiple sclerosis, and Alzheimer’s disease. Studies are generally dealing with the biological consequences of ␣ lipoic acid administration in cases associated with oxidative stress or the differences between the antioxidant activities of ␣-lipoic acid and its derivatives [32–38]. The mammary gland is a suitable model for examining its susceptibility to different carcinogenic agents because of its high cell proliferation and differentiation. Cell proliferation in the mammary gland is related to both topography of the mammary parenchyma and specific stages of the gland development that are modulated by age, hormonal variations, and parity history. The intralobular terminal duct is equivalent to the terminal ductal lobular unit in the human breast, considered the site of origin of human breast carcinomas [39–42]. Expression of proliferating cell nuclear antigen (PCNA) is elevated in the nucleus during late G1 phase immediately before the onset of DNA synthesis, becoming maximal during S-phase and declining during G2 and M phases. Its level correlates directly with rates of cellular proliferation and DNA synthesis. Aberrant cell cycle protein expression plays important pathways involved in the etiopathogenesis of breast carcinoma. Tumors with overexpression of PCNA have significantly worse outcomes [43]. Data on the efficacy and biological activity of ␣-lipoic acid on malathion mammary alterations are meager. The present study was carried out to elucidate the possible protective effect of ␣-lipoic acid treatment on malathion induced breast histopathological and biochemical alterations in rats. Moreover, the immunohistochemical changes in proliferation-apoptosis balance induced by malathion were analyzed. Materials and methods The experimental protocol was approved by the Institutional Animal Care and Use Committee of Qassim University, College of Medicine, Buridah, Qassim Region, KSA. Experimental design Forty Wistar female rats aged 39 days with average weights of 150–170 g were obtained from Qassim University Animal Facility, Faculty of Medicine, Qassim University, Buridah, KSA. They were housed in Animal facility, with room temperature maintained at 27 ◦ C, relative humidity of 50–70% and an airflow rate of 15 exchange/h. Also, a time controlled system provided 07.00–21.00 h light and 21.00–07.00 h dark cycles. All rats were given ad libitum access to Teklad rodent chow diet and water from sanitized bottle fitted with stopper and sipper tubes. Acclimatization was for 1 week before the experiment. The female rats were divided into four groups, ten animals each: Group 1: control group was injected intraperitoneally (ip) with saline solution. Group2: experimental animals were injected (ip) with 170 mg/kg body weight of malathion twice a day for five days (malathion (CAS 121-75-5) sc211768, Santa Cruz Biotechnology, Inc., USA) Purity: ≥95%. Group 3: experimental animals were orally given ␣-lipoic acid solution at a dose of 20 mg/Kg body weight, after three hours of treatment with malathion at the same dose given to group 2 (␣-lipoic acid solution (CAS 1077-28-7) sc-202032, Santa Cruz Biotechnology, Inc., USA) Purity: ≥95%. Group 4: animals were treated with ␣-lipoic acid at the same dose given to group 3. Twenty four hours and 90 days after the last treatment of animals, ten animals from each group were anaesthetized and opened by a midline incision from the pubis to the submaxillary area to remove the mammary glands. The skin was dissected to expose the six pairs of mammary glands (thoracic, abdominal and inguinal).

Please cite this article in press as: O.M. Omran, O.H. Omer, The effects of alpha-lipoic acid on breast of female albino rats exposed to malathion: Histopathological and immunohistochemical study, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.02.006

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Histopathological study

Biochemical tests

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At the end of the experiment, the mammary glands were fixed, embedded in paraffin, processed for paraffin sectioning, cut at 5–7 ␮m and treated with Harris haematoxyline and eosin (H&E) for general histopathological examination. The preparations were evaluated by means of a bright-field microscope and photographed. Furthermore, complete autopsies were performed to detect any metastases.

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Immunohistochemical study

Rat acetylcholinesterase (AChE) activity, Glutathione Peroxidase, Superoxide Dismutase, Total Antioxidant status (TAS) and Catalase were assayed using double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) commercial kits (Mybiosource, San Diego, CA, USA). Sex hormones (Estradiol, Progesterone and FSH) were similarly assayed using double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) commercial kits (Mybiosource, San Diego, CA, USA). This assay employs the quantitative sandwich enzyme immunoassay technique by ELISA kit. At the end of the experiment, all rats were fasted overnight and blood samples were collected from the rats. The animals were sacrificed under diethyl ether anesthesia, following overnight fasting, and blood sample was collected before sacrifice via intra-cardiac puncture using heparin and sodium citrate as anti-coagulants. The heparinized samples were centrifuged to separate the plasma. Then, the plasma samples were stored at −80 ◦ C.

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Immunostaining was performed for the immunohistochemical antibodies on 4-␮m thick, formalin fixed, paraffin embedded sections, using the streptavidin–biotin immunoperoxidase technique. Briefly, sections mounted on glass slides were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.6% H2 O2 . Sections were then immersed in the retrieval solution (10 mM sodium citrate buffer, pH 6.0) and subjected to heat-induced antigen retrieval for 10 min (Microwave at ∼750 W). Non-specific protein binding was blocked with 10 min exposure to 10% normal goat serum. Slides were incubated with the primary antibodies: a mouse monoclonal Anti-PCNA (PC10): sc-56 antibody (1:50 dilution; Santa Cruz Biotechnology, Inc., USA), Mouse monoclonal Anti-p53 antibody [PAb 240] (ab26) (1:50 dilution); (1:50; Abcam Company, USA) and Rabbit polyclonal Anti-Bax (B-9): sc-7480 (1:50 dilution; Santa Cruz Biotechnology, Inc., USA). After brief rinsing in phosphate buffer solution, a secondary-staining Universal Dako Labelled Streptavidin-Biotin2 System, Horseradish Peroxidase (LSAB2, System, HRP Code K0673 DAKO, USA) was used according to manufacturer instructions. All the specimens were batch-stained in the same run. Sections were next treated with peroxidase-labeled streptavidin for 30 min at room temperature and incubated with 14-diaminobenzidine for 5 min. They were counterstained with hematoxylin, dehydrated in alcohol, cleared in xylene and cover slipped. Sections were examined using Olympus BX40 Research Microscope with digital camera (Hamburg, Germany) and photographed. Results were evaluated semi-quantitatively and recorded. Similar sections running in parallel but with omission of the primary antibody were used as negative controls in each staining run. Specimens consisting of human urothelial cancer (for PCNA protein), human squamous cell carcinomas of the skin (for p53 protein) and human testis tissue (for Bax protein) were used as positive controls.

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Semi-quantitative evaluation of immunostaining

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Percentages of PCNA-immunostained nuclei (PCNA labeling index) and p53-immunostained nuclei (p53 labeling index) were calculated in each selected section for control rats, and treated rats, using the following formula: number of labeled nuclei × 100/total number (labeled + unlabeled) of nuclei. Measurements were carried out using an Olympus microscope at a magnification of 400× with random selection of fields to be studied. Tumors showing 5–10% cells with clear and unequivocal nuclear staining were identified positive cases. An average of 10 fields per section was examined, and a total of 500 epithelial nuclei were evaluated per section in each group (controls, malathion, or malathion + alpha lipoic acid) per animal [44]. The cytoplasmic expression of Bax was evaluated using an immunoreactivity score (IRS), which takes into account both the percentage of positive cells and staining intensity. This scoring method avoids the disadvantages of scoring single-positive cells or positive intensity scoring and more accurately reflects the results of immunohistochemical reactions [45].

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Statistical analysis

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Results were statistically analyzed using Statistical Package for the Social Sciences (SPSS) software program for windows, (version 13.0; SPSS Inc., Chicago, IL, USA). Statistical analysis was done using ANOVA test (analysis of variance). The results were presented as mean ± standard error of mean (SEM). The difference was considered significant at P < 0.05.

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Results

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Histopathological results

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Morphological mammary changes in rats following the administration of malathion and ˛-lipoic acid. Examination of the animals (90 days from time of malathion administration) revealed development of palpable variable sized (0.2–2.4 cm) single or multiple masses involving mammary gland tissues in 6 out of 10 of malathion treated-animals (Table 1, Fig. 1). The lesions had grayish white cut surface. Some tumors showed areas of hemorrhage and necrosis with occasional cystic changes. In malathion + ␣-lipoic acid-treated group, only a single mass was found in the mammary gland tissues in 1 out of 10 rats. It should be noted that no metastases were detected in the autopsies. Histologically, the parenchyma of the control resting mammary gland (group A) is composed of a single or clusters of widely separated patent small ducts surrounded by adipose connective tissue. There are small numbers of interlobular ducts which are lined with two layers of cuboidal cells and surrounded by dense fibrous connective tissue (Fig. 2a). In malathion treated group (group B), proliferative changes (adenosis and epitheliosis with atypical hyperplasia) were seen in all cases (Fig. 2b). Well to moderately differentiated adenocarcinoma were seen in the 6 rats (Fig. 2c). These changes were completely decreased (only one case showed adenocarcinoma) or absent in malathion + ␣-lipoic Table 1 Tumor number per rat in group B (malathion) and group C (malathion + ␣-lipoic acid).

Group B Group C

Number of rats

Number of rats with no tumors

Number of rats with tumors

Single

Multiple

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4 9

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Please cite this article in press as: O.M. Omran, O.H. Omer, The effects of alpha-lipoic acid on breast of female albino rats exposed to malathion: Histopathological and immunohistochemical study, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.02.006

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Group A Group B Group C Group D

No. of rats

Bax + cases (%)

PCNA + cases (%)

P53 + cases (%)

10 10 10 10

9 (90) 2 (20) 4 (40) 8 (80)

0 (0) 8 (80) 0 (0) 0 (0)

0 (0) 7 (70) 1 (10) 0 (0)

Group A = control; group B = malathion; group C = malathion + ␣-lipoic acid; group D = ␣-lipoic acid

Immunohistochemical results

Fig. 1. Gross features of mammary carcinoma in the malathion-treated group showing well circumscribed, rounded to oval tumor mass.

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acid-treated group (group C) with increased apoptosis and stromal desmoplastic reaction composed of admixture of lymphocytes, plasma cells, histiocytes and fibroblasts (Fig. 2d). ␣-Lipoic acid treated group (group D) showed morphological picture similar to that of the control with no pathological features (data not shown).

Compared to normal controls (group A) (Figs. 3–5a), expression of proapoptotic proteins Bax were significantly decreased by malathion treatment (group B) (Fig. 3b) (Table 2). On the contrary, we observed that malathion treatment remarkably increased the expression of the proliferation marker PCNA (group B) (Fig. 4b) and the mutant marker p53 (group B) (Fig. 5b). Malathion + ␣-lipoic acid-treated rats (group C) showed moderate expression of Bax (Fig. 3c) and absent PCNA and p53 expression similar to controls (Figs. 4c and 5c) (Table 2). These results demonstrate that ␣-lipoic acid decrease mammary alteration induced by malathion by differential regulation and activation of proteins involved in cell cycle, apoptosis and proliferation. Biochemical findings When the rats treated with malathion and/or ␣-lipoic acid were compared with the control animals at the end of the experiment,

Fig. 2. Histopathological profiles of representative mammary tissues from various experimental rat groups: (a) control animal shows normal lobular architecture (H&E); (b) malathion-treated animal shows fibrocystic changes with atypical hyperplasia (arrows) (H&E); (c) malathion-treated animal shows well differentiated adenocarcinoma (H&E,); (d) malathion plus alpha lipoic acid-treated animals (arrow mark apoptotic cell) (thick arrow mark lymphocytic infiltration) (H&E). Scale bars 100 ␮m in (a, c and d) and 200 ␮m in (b).

Please cite this article in press as: O.M. Omran, O.H. Omer, The effects of alpha-lipoic acid on breast of female albino rats exposed to malathion: Histopathological and immunohistochemical study, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.02.006

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Fig. 3. Immunohistochemical detection of proapoptotic marker Bax in mammary tissues from various experimental rat groups: (a) control animal with strong diffuse cytoplasmic bax expression; (b) malathion-treated animal with weak focal bax expression in the malignant cells (arrows); (c) malathion plus alpha lipoic acid-treated animal shows moderate diffuse bax expression. Scale bars 50 ␮m in (a) and 100 ␮m in (b and c).

Fig. 4. Immunohistochemical detection of the proliferation marker PCNA in mammary tissues from various experimental rat groups: (a) control animal shows absent nuclear PCNA expression; (b) malathion-treated animal shows increased PCNA expression in the malignant cells (arrows); (c) malathion plus alpha lipoic acid-treated animal shows absent PCNA expression similar to the control. Scale bars 100 ␮m in (a–c).

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they did not differ significantly in terms of glutathione peroxidase and total antioxidants but significantly lower in terms of superoxide dismutase and catalase (Table 3). Significant decreases in estradiol, progesterone and FSH levels were observed in malathion group compared to control. CHE in plasma was not significantly different in malathiontreated group compared to control whereas CHE in RBC was significantly lower (P < 0.001) in malathion group compared to control (Table 3). As shown in Table 3, there were significant differences between malathion group and malathion plus ␣-lipoic acid group

in catalase (P < 0.05), FSH (P < 0.05) and CHE in serum and RBC (P < 0.001).

Discussion Results obtained from the present study showed that chronic exposure to malathion produced signs of histological alterations in the breast marked by fibrocystic changes, atypical hyperplasia and malignant changes. These results suggest that chronic exposure

Fig. 5. Immunohistochemical detection of cellular tumor antigen p53 in mammary tissues. (a) Control animal shows absent nuclear p53 expression; (b) malathion-treated animal shows increased nuclear p53 expression in the malignant cells; (c) malathion plus alpha lipoic acid-treated animal shows few to absent p53 expression similar to the control. Scale bars 100 ␮m in (a–c).

Please cite this article in press as: O.M. Omran, O.H. Omer, The effects of alpha-lipoic acid on breast of female albino rats exposed to malathion: Histopathological and immunohistochemical study, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.02.006

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Table 3 Biochemical changes in rats treated with malathion and/or ␣-lipoic acid. Group A Glutathione peroxidase (U/L) SOD (U/mL) T. Antioxidants (pg/mL) Catalae (U/mL) Estradiol (pmol/L) Progesterone (ng/mL) FSH (IU/L) CHE (plasma) (nmol/L) CHE (RBC) (nmol/L)

6.31 21.3 8.67 7.37 1.22 1.56 1.22 15.7 27.7

± ± ± ± ± ± ± ± ±

3.0a 0.87a 0.47a 0.23a 0.19a 0.17a 0.07a 0.36a 1.37a

Group B 5.05 14.4 8.44 5.28 0.57 0.54 0.97 13.7 50.3

± ± ± ± ± ± ± ± ±

1.18a 0.87b 0.39a 0.29b 0.10b 0.04b 0.04b 0.68a 5.56b

Group C 5.52 11.8 9.6 3.51 0.63 0.63 0.69 8.36 29.9

± ± ± ± ± ± ± ± ±

2.02a 0.81b 0.51a 0.38c 0.03c 0.03a 0.03bc 0.33b 1.34a

Group D 4.92 15.9 7.26 5.63 1.03 1.03 1.06 13.5 34.3

± ± ± ± ± ± ± ± ±

2.87a 0.80c 0.49a 0.73b 0.07ac 0.07a 0.07a 0.94a 1.75a

Group A = control; group B = malathion; group C = malathion + ␣-lipoic acid; group D = ␣-lipoic acid. Values are mean ± S.E. a,b,c Means within rows with no common superscripts differ significantly (P < 0.05).

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to malathion can produce long-term human health consequences. Numerous studies in humans have indicated that there is an elevated risk of developing cancers such as non-Hodgkins lymphoma and leukemia in farmers exposed to malathion and other chemicals used in agriculture [46–48]. Moreover, a recent study showed that malathion is a specific pesticide which is implicated as risk factor for aggressive prostate cancer [49]. Our results showed that 60% of rats exposed to malathion showed neoplastic transformation in the epithelium of ducts while they were in the process of development. These results indicated that the proliferative changes observed in such structures may have induced the formation of mammary ductal carcinomas. Previous studies demonstrated that malathion induced mammary tumors in 24% of 44 day-old treated rats [2,13]. In contrast, to potent carcinogens, which induced mammary carcinomas in 100% of intact females Sprague-Dawley rats by chemical carcinogens, such as dimethylbenz[␣]anthracene (DMBA), organophosphorous pesticides seem to have a slow and less infiltrating effects [39]. Like other organophosphate insecticides, purported mechanisms of action include direct genotoxicity of malathion and potential endocrine disruption [50–52]. The genotoxic effect of malathion is mediated through oxidative stress as evidenced by a significant production of malondialdehyde (MDA), an end product of lipid peroxidation [53,54]. Experimental evidence showed that pesticides may modify gene promoter DNA methylation levels, suggesting that epigenetic mechanisms may contribute to pesticide-induced carcinogenesis [55]. Results obtained from the present study demonstrated significant decrease in the proapoptotic protein Bax with malathion treatment that was restored by alpha lipoic acid treatment. Expression of Bcl-2 family proteins, such as Bax, induces mitochondrial apoptosis and its expression is reduced in several types of cancers [56]. ␣-Lipoic acid (LPA) has been reported to scavenge a variety of oxygen species [57–59]. The natural antioxidant alpha-lipoic acid induces apoptosis in MCF-7 human breast cancer cells [59]. The reduction in mitochondrial proapoptotic stimuli is indicative of autophagic process inducing cytoprotective effects in the early stage of stress. Down regulation of apoptotic signaling may be due to reduction in ATP and ROS, and genotoxic potential of malathion [60]. Our study demonstrated significant increase in the proliferating cell nuclear antigen (PCNA) and mutant p53 expression with malathion treatment that was suppressed by alpha lipoic acid treatment. These results was in agreement with other study that reported that malathion increased PCNA and induced mutant p53 protein expression of MCF7 cells in comparison to controls which indicated that organophosphorous pesticides can induce more changes in the malignant breast cell line, inducing another step in the progression of the transformation process [61]. In unstressed cells, p53 levels are kept low through a continuous degradation of p53. The TP53 gene is damaged, tumor suppression is severely

reduced. More than 50% of human tumors contain a mutation or deletion of the TP53 gene 39 [62]. A previous study demonstrated significantly increased the number of ducts in stage of proliferation after 10 and 20 days of malathion treatment, which was coincident with the increase in mutant p53 protein expression by western blot analysis as quantified by the relative unit of densitometry. The organophosphorous pesticides malathion induced malignant transformation of breast cells through genomic instability altering p53, considered pivotal to cancer process [12]. p53 is frequently mutated in tumor cells, and mutant p53 is often highly expressed due to its increased half-life. Thus, targeting mutant p53 for degradation might be explored as a therapeutic strategy to manage tumors that are addicted to mutant p53 for survival [63]. Alpha lipoic acid inhibits cell proliferation of tumor cells in vitro and in vivo. It reduced cell viability/proliferation and increased apoptosis in all investigated cell lines [57,58]. In the mouse xenograft model with malignant cells, daily treatment with alpha lipoic acid retarded tumor progression [57]. Moreover, alpha lipoic acid induced apoptosis in hepatoma cells suggesting its usefulness in liver cancer therapy [64]. The biochemical parameters were investigated in this study to assess the metabolic parameters, antioxidant stress and hormonal profile changes in rats exposed to malathion and to elucidate the possible protective effect of ␣-lipoic acid on malathion induced biochemical alterations in rats. No significant differences in Glutathione peroxidase and total antioxidants were observed between all groups at the end of the experiment. Whether or not there was a transient increase in those two parameters at an earlier time remains to be elucidated. The activity of catalase and superoxide mutase in the malathiontreated animals were significantly decreased when compared to control (P < 0.001 and P < 0.01, respectively). Treatment with ␣lipoic acid significantly prevented decrease in the catalase activity compared to the malathion treated rats (P < 0.05). Treatment with ␣-lipoic acid insignificantly elevated the SOD levels (P > 0.05) as compared to the malathion-treated animals. This data was represented in Table 3. The results of this study which reveals significant decreases in the activities of the antioxidant enzymes (CAT, SOD) suggest that exposure to malathion can result in free radical generation. These results came at agreement to Selmi et al. but at variance to the findings of El-Gamal et al. [65,66]. It should be stressed that oxidative stress results in reduction in tissue antioxidants because these agent are utilized in terminating the lipid peroxidation chain reactions. The decrease of Estradiol, Progesterone and FSH in malathiontreated animals is not surprising. Earlier reports have showed that the level of 17-␤-estradiol and progesterone concentration in malathion-treated Tilapia Fish females, were significantly decreased [67]. The reduction in plasma 17-␤-estradiol and progesterone could be explained by the inhibitory effects of malathion on

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the secretion of pituitary gonadotrophins (FSH and LH) [68]. It may be of relevance to mention that Uzuna et al. reported significantly lower plasma FSH, LH and testosterone levels in malathion-treated rats than the control rats [51]. The inhibition of red blood cell cholinesterase (RBC ChE) was much higher than that of plasma cholinesterase (PChE). The most sensitive endpoint in short-term studies is inhibition of AChE activity, with RBC AChE being more sensitive than plasma cholinesterase (PChE) because RBC AChE is slower to depress, slower to recover. Erythrocyte AChE inhibition seemed to be relieved in malathion + ␣-lipoic acid rats [69]. In consistence with our results, a previous study showed that the administration of ␣-lipoic acid before malathion exposure to rat can prevent severe hematobiochemical parameters changes [70]. The induced malathion tumors in this study were locally aggressive but no single case of metastasis was detected in the autopsies which open the door for speculation. A new study reported that tumors appearing histologically malignant in the rat have features in common with the intraductal and infiltrating ductal carcinomas in humans, but few spontaneously metastasize and most of the lesions found in the rat mammary glands have their counterpart in human pathology [71]. Our study demonstrated that rats treated with alpha lipoic acid in association with malathion showed absent or small size mammary tumor, stimulated apoptosis, and exhibited stromal desmoplastic reaction (signs of tumor regression). The immune response in the tumor microenvironment is complex, consisting of cells from both the adaptive and innate immune systems. The phenotype and function of these cells are dictated by cytokines present in the microenvironment, as well as by the interactions of these cells with the tumor cells and each other [72]. The appearance of abundant apoptotic cells and increased Bax expression in cells of malathion + alpha lipoic acid group suggests the role of apoptosis as one of the mechanisms of antipoliferative effects of alpha lipoic acid. These observations could be helpful in exploring effects of cancer therapeutic agents in intratumoral region. As compared to the control group, malathion-treated group showed development of proliferative and malignant breast changes that was associated with decreased expression of Bax and increased expression of PCNA and p53. Alternatively, decrease of these histopathological changes in ␣-lipoic acid-treated group was associated with increased expression of apoptotic activity marker Bax and decreased or absent expression of PCNA and p53 similar to the control. Results of the current study clearly demonstrated the following observations. First, administration of malathion was associated with development of benign proliferative breast changes, atypical hyperplasia and malignant changes as well as a decrease in apoptotic activity (Bax). In contrast, administration of ␣ lipoic acid was associated with decrease or absent expression of markers of proliferation (PCNA) and tumorigenicity (p53).

Conclusions Alpha lipoic acid exhibited a striking reduction of malathioninduced mammary tumor incidence, and reversed intra-tumor histopathological alterations. Alpha lipoic acid suppressed the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and mutant p53 expression, induced apoptosis confirmed by upregulation of the proapoptotic protein Bax. Our results clearly provide the first experimental evidence that Alpha lipoic acid exerts chemopreventive effect in the breast malignant changes by suppressing abnormal cell proliferation and inducing apoptosis with an oncostatic effects during an early-stage breast cancer. Therefore, alpha lipoic acid with its protective antioxidant

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effects seems to be a promising compound for cancer treatment and encourage further studies to explore full potential of alpha lipoic acid as breast cancer chemotherapeutic agents.

Acknowledgments Authors acknowledge the financial support of this study by King Abdulaziz City for Science and Technology (KACST)-KSA. Small Q4 Project Grant # 33-13.

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