Quantification and digestion of testicular DNA in rats ...

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Venkataswamy Mallepogu, Divya Kurumala, Kamala Katepogu,. Thyagaraju Kedam. Department of Biochemistry, Sri Venkateswara University, Tirupathi, ...
Quantification and digestion of testicular DNA in rats under the influence of acrylamide Lakshmi Narasaiah Uppalapati, Suman Bukke, Meena Bai Malekar, Venkataswamy Mallepogu, Divya Kurumala, Kamala Katepogu, Thyagaraju Kedam Department of Biochemistry, Sri Venkateswara University, Tirupathi, Andhra Pradesh, 517502, India, Email: [email protected]

ABSTRACT The increased releasing of harmful chemicals to our life, results in adverse outcomes in the survival and health of off springs. The epidemiologic evidence associated with of male exposure to exogenous agents suggests abnormal reproductive outcomes, such as birth defects, foetus loss, abortion, etc. Acrylamide (AA), an industrial chemical, is used in waste water treatment, adhesives and cosmetics, and also in laboratories. It is genotoxic, in in vitro and in vivo, in both somatic and germ cells. The male wistar rat testis treated with different doses of AA, at different intervals of time has demonstrated the ability of AA on the DNA damage. In in vivo on the restriction endonuclease digestion analysis (Bam HI and Hind III) on DNA, AA was found to possess strand weakening and breaking properties in rat testicular cells. At least 7.4kb of 5’ DNA may be present at upstream of the selected 21.13kb genomic DNA of testicular tissue may undergo degradation to get 200bp sequences. This result also suggests that it may participate in linker sequences of nucleosomal DNA for modification and may serve as an agent for induction of degradation. Therefore our study made on quantification and restriction digestion analysis on testicular DNA has revealed that the degree of heavy genotoxicity effect by acrylamide is dose and time dependent on rat’s reproductive system. Keywords: acrylamide, alkalization, rat testis, DNA damage, restriction digestion

INTRODUCTION In today’s modernization in society, we are constantly being exposed to an innumerable number of potentially harmful chemicals in our life from our recipes and environment. The reproductive cells are of particular concern since they pass genetic material to next generations. In mammalian systems sperm chromatin compact six times and this packaging is useful for several reasons, i.e. to reduce the volume of spermatozoa, minimise the damage by exogenous agents before fertilization and keep the genome transcriptionally inactive than the metaphase chromosomes [1]. Among the fertile couples, the sperm already selected through apoptotic pathways can compete to fertilize with a single ovum. The healthiest sperm (with intact DNA) will fertilize the ovum; sperm with damaged DNA may accomplish fertilization shows poor pregnancy [2,3]. Intracytoplasmic sperm injection and in vitro fertilization (IVF) make it more likely that sperm with damaged DNA will fertilize the egg [4-6]. Thus, an evaluation about sperm nuclear DNA damage and apoptosis is of clinical importance [7]. The sperm has several major advantages compared to reproductive cells; most importantly the male reproductive system is constantly producing sperm through out the life span of an individual. The mature sperm do not have DNA repair capacity and three potential mechanisms independently or co-independently accountable for basic sperm damage [8] have been identified, i.e. defective chromatin condensation during spermatogenesis [9-11]. Apoptotic events during Research Article, Biotechnol. Bioinf. Bioeng. 2012, 2(1):584-590 © 2012 Society for Applied Biotechnology; pISSN 2249-9075, eISSN 2249-9938

Biotechnol. Bioinf. Bioeng. 2012, 2(1):584-590

spermatogensis, epididymal maturation or in the ejaculate reactive oxygen species [12,13] can form due to oxidative stress. Many toxicants do not cause strand breaks but damage the DNA by cross linking [14]. In sperm, such highly damaged cells, showing extensive DNA fragmentation cannot be excluded [15]. DNA fragmentation to low molecular weight is associated generally with late apoptotic events [16]. The acrylamide on rats can effect on various reproductive parameters including decreased sperm count and increased abnormal sperm morphology [17], testicular damages such as vacolulation severe swelling of the round spermatid and DNA breakage during specific germ cells stages [18]. Male rats administered acrylamide significantly reduces in mating capacity, fertility and transport of sperm in uterus. However no effect was found on female rats [19]. Therefore to elucidate the effect of acrylamide on the sperm DNA the research was conducted and the results were discussed.

MATERIALS AND METHODS The male wistar rats weighed about 150-200 gms with an age of three months old were purchased from Sri Venkateswara Enterprises (Animal Agency), Bangalore, India. These rats were acclimated for seven days after arrival from the supplier (control and treatment groups consisted of six animals each). Temperature was maintained at 22°C with relative humidity of 40-50% on 13L:11D hrs (5 am - 5 pm) cycle. Animals were housed individually in polycarbonate cages and provided food (Purina Certified Rodent Chow 5002 and tap water ad libitum). The animals were treated with different doses of acrylamide for each group. Collection of blood at different intervals of time was made. Testes cauda epididymus of all rats were isolated further for experimentation after decapitation of animal. The tissues were washed with Phosphate Buffered Saline (PBS) and homogenized in lysis buffer. After centrifugation for 5 mins at 1600Xg the supernatants were collected and the extraction procedure was repeated with the same amount of lysis buffer. The supernatant was brought to 1% SDS and treated for 2hrs with RNase at 56°C followed by digestion with proteinase for at least 2hrs at 27°C. After addition of half volume of ammonium acetate the DNA was precipitated with 2.5 volume of absolute ethanol and centrifuged at 1600Xg. Alcohol was drained by inverting the tubes on blotting paper then the pellet was dissolved in TE buffer, pH 8.4. Analysis of UV absorption of nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. The DNA absorption was measured at 260nm and DNA content was recorded using the calculation as, concentration of DNA (µg/ml) =OD260 × dilution factor. A ratio 1.8-2.0 denotes that the absorption in the UV range is due to pure nucleic acids. Ratios lower than the 1.8 indicates the presence of proteins and or other UV absorbance. A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or phenol. Apoptotic genomic DNA was fractionated on 0.9% agarose gel electrophoresis [20]; about 15µl of the sample with 5 µl of the loading dye and marker DNA were loaded carefully in to the respective wells without disturbing the gel. After conducting electrophoresis at 50 mV, agarose gel photographed with gel doc. Analysis of DNA molecules is often hampered by their large size. One way of generating specific smaller DNA fragments from larger ones through the use of restriction enzymes, which are sequence specific DNA endonucleases. Two restriction enzymes were used to generate fragments of DNA are, Bam HI - 5´GGATCC3´ and Hind III- 5´AAGCTT3´. Each group was set up to generate the following restriction digests, in a 0.5ml micro centrifuge tube, as mentioned in table 1. Thawed DNA samples were kept on ice by the addition of 2µl of RE buffer, 1mg/1ml of BSA (1 µg/1 µl), 12µl of DNA samples (21.13kb) and 200µg/ml RNase (0.2µg/µl) were transferred in to series of Eppendorf tubes and all tubes were made up to 20µl of final volume with 2µl of restriction enzyme, the contents were mixed for few minutes by using pipette and incubated at 37°C for overnight. After overnight incubation, tubes were transferred to 70°C water

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bath for 10 mins to inactivate restriction enzymes. The restriction digests were stored at -20°C as 4 µl aliquots of each reaction mixture after mixing with 16µl of loading buffer and analyzed by agarose electrophoresis.

RESULTS AND DISUSSION Determination of DNA in testicular tissue The DNA isolated from control and acrylamide induced rat testicular tissues contained 59.60µg/gm of testicular tissue. In positive control DNA content was reduced to fatal level, i.e. 17.13µg/gm of tissue. The rats treated with acrylamide at different concentrations showed the decrease of DNA based on the concentration of acrylamide increase (Table 2). In 16, 32, 48, 64, 80 and 96 mg of treatment of AA, the decrease of DNA was 9.87, 11.14, 23.27, 26.87, 26.67 and 41.87µg respectively, observed upon acrylamide treatment in rats testis.

Fractionation of DNA Genomic DNA isolated from testis sample was fractionated using 0.9% agarose gel electrophoresis [23]. Agarose gel photograph showed that the degree of variations of DNA was decreased as the concentration of acrylamide was increased (Figure 1).

λ DNA Hind III Digest

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Figure 1. Electrophoresis analysis of DNA of control and acrylamide treated rats testicular DNA. Lane 1: λ DNA, Hind III digest marker, Lane 2: positive control (8 mg CPA), Lane 3: 96 mg AA treated rat testicular DNA, Lane 4: 80 mg AA, Lane 5: 64 mg AA, Lane 6: 48 mg AA, Lane 7: 32 mg AA, Lane 8: 16 mg AA, Lane 9: vehicle control (saline).

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Table 1. Contents for preparation of DNA digest by restriction digestion. Digest 1 2

DNA(1.73 µg/1 µL) 12 µL 12 µL

10X enzyme buffer 2 µL buffer E 2 µL buffer E

1µg/µL BSA (1µg/µl) 2 µL 2 µL

RNase (0.2 µg/µl) 2 µL 2 µL

Restriction enzyme 2 µL Bam HI 2 µL Hind III

Table 2. Effect of acrylamide on testicular DNA content. Groups (mg AA)

Mean±SE* µg/gm of tissue

Control Positive control 16 32 48 64 80 96

59.60±1.52 17.13±2.84 49.73±0.66 48.46±1.45 33.66±1.20 32.73±0.88 29.93±0.88 17.73±0.88

Decrease in concentration of DNA with control (µg/gm of tissue) 42.47 09.87 11.14 23.27 26.87 29.67 41.87

Fold decrease in DNA concentration 3.48 1.20 1.23 1.77 1.82 1.99 3.36

Restriction digestion analysis In the present study, the DNA fragment, 21.13kb, of size showed in figure 1 was electroeluted and all fragments were digested with restriction endonucleases, Bam HI and Hind III, separately, and their DNA trailing patterns were analyzed by agarose gel electrophoresis using Gene ruler DNA ladder mix (Fermentas, USA) as a marker (Figure 2 and 3). In vehicle control group restriction banding patterns showed no trailing of DNA that reveals the intact nature of the control group DNA. The cyclophosphamide treated positive control group DNA showed more trailing than any other groups. In acrylamide treated groups at different concentrations as shown progressive trailing of restriction digested DNA, but at higher concentrations (64, 80 and 96 mg) were high degree of variation in DNA band trailing patterns reveals critical DNA damage treatment (64 mg). The degree of trailing of DNA is the phenomenal damage [20] of DNA that is clearly observed in Bam HI and Hind III restriction digestion analysis. Figure 2 and 3 shows a comparative restriction fragments patterns of controls and acrylamide treated testicular DNA digested with Bam HI and Hind III enzymes. Male sterility, infertility and abnormal progeny outcome are some of the consequences resulting from the exposure of germ cells to stress such as environmental chemicals and drugs [21]. Testicular cancer is a disease in which cells become malignant in one or both testicles. Testicles produce and store sperm and are the body’s main sources of male hormones. These hormones control the development of reproductive organs and male characteristics. The results of the animal toxicity studies indicate that acrylamide is carcinogen in rodents and produces toxic effects on reproductive and nervous systems [22]. Based on the results of acute toxicity studies, the oral LD50 value for acrylamide is ~100 to 150 mg/kg of body weight in mice, rabbits, guinea pigs and rat [2324]. Acrylamide has been evaluated for reproductive toxicity in multigenerational studies in rats and mice. At higher doses in both rats and mice, acrylamide has resulted in decrease in fertility, in addition to changes in pre- and post implantation loses [25,26]. In contrast to such potent carcinogenic substances, genotoxic effects have been described for acrylamide mutagenesis or genotoxic effects now only in very high concentration in the laboratory animals. In human keratinocytes (HaCaT cell lines) the acrylamide show damage to DNA as revealed by comet assay tailing [27].

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500bp 400bp 300bp 200bp 100bp Figure 2. Bam HI restriction digestion analysis of DNA fragment (21.13kb) of electro eluted controls and acrylamide treated rats testicular DNA. Lane 1 and 10 - marker (Fermentas, USA), Lane 2: positive control (8 mg CPA), Lane 3: vehicle control (saline), Lane 4: 16 mg AA, Lane 5: 32 mg AA, Lane 6: 48 mg AA, Lane 7: 64 mg AA, Lane 8: 80 mg AA, Lane 9: 96 mg AA treated rat testicular DNA.

The present study demonstrated the ability of AA to cause DNA damage in vivo by the restriction endonucleases digestion analysis, Bam HI and Hind III. Previous work has demonstrated that DNA damage was recorded in blood, brain, bone marrow, liver, testes and adrenal glands of rats, 24 hrs after treatment with single doses of 18, 36 or 54 mg/kg body weight [28]. AA has been reported to possess clastogenic and mutagenic properties in vivo. However, AA itself is known to react quite slowly with DNA [29]. Therefore, its metabolic GA, formed by CYP2E1, is proposed to represent the ultimate carcinogen and mutagen in vitro and in vivo [30,31]. One critical point in the previous cell culture experiments was the question whether the culture conditions might lead to a rapid loss of CYP2E1 activity in primary rat hepatocytes, prohibiting the effective conservation of AA to the more reactive GA [32]. Therefore, we aimed to study the DNA breaking effect of AA in vivo by using rat system at different concentrations of AA and cyclophosphamide drug as a positive control. AA treated groups when compared with control have showed that the concentration of apoptotic DNA is decreased as the concentration of AA increased and it is augmented at the range of 64 mg of AA as a two fold decrease. The restriction digestion analysis of 21.13 kb range

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fragments of electroeluted apoptotic DNA of treated groups with Bam HI and Hind III have also supported with its high degree of variation in their gel patterns (Figure 4). AA was found to possess strand breaking properties in rat testicular cells and in vivo DNA damage was induced by AA despite substantial expression of CYP2 [32].

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1000bp 500bp 400bp Figure 3. Hind III restriction digestion analysis of DNA fragment (21.13 kb) of electro eluted controls and acrylamide treated rats testicular DNA. Lane 1 and 10 - marker (Fermentas, USA), Lane 2: positive control (8 mg CPA), Lane 3: vehicle control (saline), Lane 4: 16 mg AA, Lane 5: 32 mg AA, Lane 6: 48 mg AA, Lane 7: 64 mg AA, Lane 8: 80 mg AA, Lane 9: 96 mg AA treated rat testicular DNA.

Figure 4. Restriction map of rat testicular DNA (21.13Kb).

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From our studies, it is expected that 21.13Kb segment can affect the DNA to degrade to 200bp (nucleosome). The digestion analysis has revealed that acrylamide can facilitate sites of restriction enzymes to degrade the DNA and cause damage to cell viability. Hence as we know from food science and cooling studies, the fried food can get most of acrylamide content and that may enhance damage to cell, cell viability and DNA degradation.

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