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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (12): 1433-1442.doi: 10.3736/jcim20121216

• Original Experimental Research • Previous Articles     Next Articles

Dihydroxy-isosteviol methyl ester of Pulsatilla nigricans extract reduces arsenic-induced DNA damage in testis cells of male mice: Its toxicity, drug-DNA interaction and signaling cascades

Samadder Asmita1,Das Jayeeta1,Das Sreemanti1,Das Durba1,De Arnab1,Bhadra Kakali2,Rahman Khuda-Bukhsh Anisur1()   

  1. 1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
    2. Entomology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
  • Received:2012-05-17 Accepted:2012-06-19 Online:2012-12-20 Published:2018-12-15

Objective: To evaluate the ameliorative efficacy of dihydroxy-isosteviol methyl ester (DIME) of Pulsatilla nigricans extract in arsenic-induced DNA damage in testis cells of mice.
Methods: The mice were treated with sodium arsenite (SA) solution intragastrically at a dose of 20 mg/kg per day and examined at 30, 60, and 90 d after treatment. We divided SA-intoxicated mice into two sub-groups: one fed with DIME at a dose of 35 mg/kg and the other with 85% alcohol. We analyzed the expressions of apoptotic signal proteins like CYP1A1, p53 and caspase 3, ascertained the level of cellular and DNA damage and estimated the level of testicular-toxicity biomarkers. We studied the interaction of DIME with calf thymus DNA as target through circular dichroism spectra and melting temperature profiles.
Results: We observed an elevation in all apoptotic and toxicity biomarkers leading to cellular and DNA damage in the SA-intoxicated mice which showed significant inhibition or reversal on administration of DIME. Results also showed that DIME interacted with DNA, bringing in discernible changes in structure and conformation.
Conclusion: DIME has potentials for therapeutic use in amelioration of arsenic-induced reproductive toxicity.

Key words: Pulsatilla, plant extracts, arsenites, apoptosis, spermatids, mice

Figure 1

Probable chemical structure of dihydroxy-isosteviol methyl ester."

Figure 2

Bar diagrams of activities of ACP and ALP at different time intervals Data are presented as mean±standard error of mean, n=3; *P<0.05, **P<0.01, vs SA plus placebo-treated group. ACP: acid phosphatase; ALP: alkaline phosphatase; SA: sodium arsenite; DIME: dihydroxy-isosteviol methyl ester."

Figure 3

Bar diagrams showing sperm count at different intervals of treatment Data are presented as mean±standard error of mean, n=3; *P<0.05, **P<0.01, vs SA plus placebo-treated group. SA: sodium arsenite; DIME: dihydroxy-isosteviol methyl ester."

Figure 4

Bar diagrams showing arsenic content in testis at different time intervals Data are presented as mean±standard error of mean, n=3; *P<0.05, **P<0.01, vs SA plus placebo-treated group. SA: sodium arsenite; DIME: dihydroxy-isosteviol methyl ester."

Figure 5

Images showing sperm head morphology (Light microscopy, ×200) A: Normal; B-D: SA-intoxicated (30, 60 and 90 d, respectively); E-G: SA plus placebo-treated (30, 60 and 90 d, respectively); H-J: SA plus DIME-treated (30, 60 and 90 d, respectively). SA: sodium arsenite; DIME: dihydroxy-isosteviol methyl ester."

Figure 6

DNA fragmentation assessment Ln1: Normal; Ln2: SA-intoxicated; Ln3: SA plus placebo-treated; Ln4: SA plus DIME-treated. SA: sodium arsenite; DIME: dihydroxy-isosteviol methyl ester."

Figure 7

CD spectra of calf-thymus DNA in the absence and presence of DIME CD: circular dichroism; DIME: dihydroxy-isosteviol methyl ester."

Figure 8

Graphs of DNA thermal denaturation DIME: dihydroxy-isosteviol methyl ester; OD: optical density."

Figure 9

Immunohistochemical staining for p53 in testes (Light microscopy, ×200) A: Normal; B-D: SA-intoxicated (30, 60 and 90 d, respectively); E-G: SA plus placebo-treated (30, 60 and 90 d, respectively); H-J: SA plus DIME-treated (30, 60 and 90 d, respectively). SA: sodium arsenite; DIME: dihydroxy-isosteviol methyl ester."

Figure 10

Photographs of sperm cells stained with DAPI (Fluorescence microscopy, ×200) A: Normal; B-D: SA-intoxicated (30, 60 and 90 d, respectively); E-G: SA plus placebo-treated (30, 60 and 90 d, respectively); H-J: SA plus DIME-treated (30, 60 and 90 d, respectively). DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride; SA: sodium arsenite; DIME: dihydroxy-isosteviol methyl ester."

Figure 11

Bands obtained in Western blot and RT-PCR analyses A: Western blot; B: RT-PCR. G3PDH: glyceraldehyde-3-phosphate dehydrogenase; RT-PCR: reverse transcriptase-polymerase chain reaction."

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