Number Of Micronucleated Polychromatic Erythrocytes Research Articles

Citicoline Protects against Tramadol-Induced Oxidative Stress and Organ Damage

Tramadol is a common drug of abuse which has been shown to cause neurodegeneration in the rat brain. Cytidine-5'-diphosphocholine or citicoline is an intermediate in the synthesis of phosphatidylcholine and is in use in humans for the treatment of several brain pathologies. In this study, we aimed to investigate the effect of citicoline on oxidative stress and tissue injury caused by tramadol, an opioid drug. Rats were treated with tramadol at 30 mg/kg alone or in combination with citicoline at 50, 100, or 200 mg/kg orally, once a day, for 10 days. Other groups were treated with only 0.9% saline or only citicoline at 200 mg/kg. Lipid peroxidation (malondialdehyde), nitric oxide, reduced glutathione (GSH), and paraoxonase-1 (PON-1) activity were measured in the serum. Bone marrow DNA fragmentation assay and micronucleus test were also done. In addition, histopathological examination of the brain, liver, and kidney, and immunohistochemical staining for glial cell acidic fibrillary protein (GFAP) in the cerebral cortex were performed. Results indicated that compared to the saline-treated group, repeated tramadol administration led to significant increases in serum malondialdehyde and nitric oxide concentrations by 50.0% and 70.0%, respectively. There was also a decline in GSH content and PON-1 activity in the serum by 26.3% and 51.4%, respectively. Tramadol caused marked DNA fragmentation and increased the number of micronucleated polychromatic erythrocytes (MnPCE) in bone marrow by 192.8% and 876.3%, respectively, compared with the corresponding saline control values. Histopathological studies revealed neuronal degeneration (acidophilic cytoplasm and dark nuclei) and decreased GFAP immunostaining in the cerebral cortex of tramadol-treated rats. The liver exhibited fibrosis, apoptotic hepatocytes, and inflammatory cell infiltration. Vacuolar degeneration of the tubular lining epithelium and edema of glomeruli were observed in the kidney. Citicoline administered to saline-treated rats at a dose of 200 mg/kg showed no significant effect on serum malondialdehyde, nitric oxide, GSH concentrations, or PON-1 activity compared with the saline control group. Citicoline by itself had no effect on DNA fragmentation or the number of MnPCE in the bone marrow. In tramadol-treated rats, however, citicoline (50–200 mg/kg) resulted in a significantly decreased malondialdehyde by 23.8%–31.6%. Nitric oxide decreased by 29.2%–36.2% after citicoline at 50–200 mg/kg. There was also a significant increase in both GSH by 19.6%–33.6% and in PON-1 activity by 54.8%–125.7%. In addition, citicoline caused a significant decrease in DNA fragmentation (by 29.2%–52.4%) and the number of MnPCE in the bone marrow (by 20.5%–59.5%) in tramadol-treated rats. Histopathological changes caused by tramadol in the brain, liver, and kidney were ameliorated by treatment with citicoline. We conclude that citicoline treatment results in decreased lipid peroxidation and nitric oxide along with increased GSH content and PON-1 activity in the serum of rats treated with tramadol. These changes might account for the beneficial effect of citicoline in decreasing DNA fragmentation and tissue injury caused by tramadol.

Modulation of hexavalent chromium-induced genotoxic damage in peripheral blood of mice by epigallocatechin-3-gallate (EGCG) and its relationship to the apoptotic activity

ABSTRACTThis study was conducted to investigate the relationship between modulation of genotoxic damage and apoptotic activity in Hsd:ICR male mice treated with (–)-epigallocatechin-3-gallate (EGCG) and hexavalent chromium [Cr(VI)]. Four groups of 5 mice each were treated with (i) control vehicle only, (ii) EGCG (10 mg/kg) by gavage, (iii) Cr(VI) (20 mg/kg of CrO3) intraperitoneally (ip), and (iv) EGCG in addition to CrO3 (EGCG-CrO3). Genotoxic damage was evaluated by examining presence of micronucleated polychromatic erythrocytes (MN-PCE) obtained from peripheral blood of the caudal vein at 0, 24, 48, and 72 h after treatment. Induction of apoptosis and cell viability were assessed by differential acridine orange/ethidium bromide (AO/EB) staining. EGCG treatment produced no significant changes in frequency of MN-PCE. However, CrO3 treatment significantly increased number of MN-PCE at 24 and 48 h post injection. Treatment with EGCG prior to CrO3 injection decreased number of MN-PCE compared to CrO3 alone. The MN-PCE reduction was greater than when EGCG was administered ip. The frequency of early apoptotic cells was elevated at 48 h following EGCG, CrO3, or EGCG-CrO3 exposure, with highest levels observed in the combined treatment group, while the frequencies of late apoptotic cells and necrotic cells were increased only in EGCG-CrO3 exposure. Our findings support the view that EGCG is protective against genotoxic damage induced by Cr(VI) and that apoptosis may contribute to elimination of DNA-damaged cells (MN-PCE) when EGCG was administered prior to CrO3. Further, it was found that the route of administration of EGCG plays an important role in protection against CrO3-induced genotoxic damage.

Protective Effect of Grapefruit Juice on the Teratogenic and Genotoxic Damage Induced by Cadmium in Mice

In the present study, we injected pregnant mice at Day 7 of gestation with cadmium chloride (CC) (1.5 mg/kg) intraperitoneally and determined its effect on the frequency of fetal malformations at Day 17 of pregnancy. On the same day, we also determined the level of micronucleated polychromatic erythrocytes (MNPEs) and of micronucleated normochromatic erythrocytes (MNNEs) in blood cells of both the mothers and their fetuses. A significant increase in the number of malformations was found, mainly exencephaly, micrognathia, ablephary, microphthalmia, and clubfoot, as well as a significant increase in the amount of MNPEs and MNNEs. In addition, pregnant mice were administered grapefruit juice (GJ) orally from Days 0 to 17 of the experiment (from 200 to 800 μL/g) to evaluate the potential of the juice in preventing the damage induced by CC. We found a dose-dependent decrease in the number of visceral and skeletal malformations, as well as in the number of MNPEs and MNNEs, in both the mothers and their fetuses. Furthermore, we determined the level of DNA oxidation by measuring levels of the adduct 8-hydroxy-2'-deoxyguanosine, and we found a significant increase in such level induced by CC; in contrast, there was a significant decrease when we added GJ. Therefore, the observed teratogenic and genotoxic protection can probably be related with the antioxidant potential of GJ.

Evidence That Oxymorphone-Induced Increases in Micronuclei Occur Secondary to Hyperthermia

Oxymorphone is a potent opioid analgesic. Oral administration of oxymorphone to rats at doses >or= 20 mg/kg and mice at 500 mg/kg produced an increase in micronucleated polychromatic erythrocytes (MPCEs). Oxymorphone does not produce chromosome aberrations in vitro, suggesting that the increased MPCEs in vivo may involve indirect mechanisms. Opioids are known to affect thermoregulatory mechanisms. Changes in body temperature can increase the incidence of MPCEs in rodents. Studies were conducted to examine the relationship between increased MPCEs in rats given oxymorphone and changes in body temperature. Single oral doses of oxymorphone associated with increased MPCEs (20 and 40 mg/kg) also produced a marked, rapid increase in body temperature. When animals were pretreated with sodium salicylate, peak body temperature was lower and returned to baseline more quickly than when oxymorphone was given alone. MPCEs were evaluated in rats after administration of oxymorphone (40 mg/kg) alone or following pretreatment with an oral dose of sodium salicylate. Oxymorphone alone produced a statistically significant increase in the incidence of MPCEs (3.6 per 1000 polychromatic erythrocytes vs. 0.4 in controls). The number of MPCEs in animals pretreated with sodium salicylate was similar to controls. Sodium salicylate alone had no effect on the number of MPCEs. Systemic oxymorphone exposure was not affected by sodium salicylate pretreatment; maximum plasma concentration (C(max)) and area-under-the-curve values were similar after administration of oxymorphone alone or following pretreatment with sodium salicylate. These results indicate that the increased incidence of MPCEs following oxymorphone administration is directly related to increased body temperature.

Nuclear abnormalities in erythrocytes of parrots (Aratinga canicularis) related to genotoxic damage

Nuclear abnormalities in erythrocytes, as micronuclei and nuclear buds (BE), are considered potential biomarkers of genotoxic exposure. We described previously the frequency of spontaneous micronucleated erythrocytes (MNE) in the species Aratinga canicularis. Here, we have used this species to evaluate the induction of MNE and BE by mitomycin-C. Animals were given a single intracoelomic injection of 0, 2, 3 or 4 mg/kg mitomycin-C on two consecutive days. A drop of blood was obtained after 0, 24, 48 and 72 h, and stained smears were used to count micronucleated polychromatic erythrocytes (MNPCE) and polychromatic erythrocytes with buds (BPCE)/1000 polychromatic erythrocytes. The number of MNE and BE in 10 000 total erythrocytes was also counted. MNPCE and BPCE frequencies were elevated at 24, 48, and 72 h after the administration of the lower dose (P<0.03). At a 3 mg/kg dose, the frequency of MNPCE increased at 48 and 72 h (P<0.04) whereas the number of BPCE increased, but not significantly. Administration of 4 mg/kg mitomycin-C increased the number of MNE observed at 72 h (P<0.03), the number of MNPCE at 48 h (P<0.01) and 72 h (P<0.006), the BE frequency at 72 h (P<0.05), and the frequency of BPCE at 48 and 72 h (P<0.001). While mitomycin-C appears to produce a parallel increase in MNPCE and BPCE frequencies, the MNE seemed to be a more sensitive indicator of genotoxicity than the BE. This suggests that evaluating BE and MNE in routine haematological analysis should be considered to evaluate environmental genotoxic exposure.

Chemoprotective Effect of Phyllanthus maderaspatensis. in Modulating Cisplatin-Induced Nephrotoxicity and Genotoxicity

The chemoprotective effect of Phyllanthus maderaspatensis. Linn (PME) was studied on cisplatin-induced nephro- and genotoxicity in male Swiss albino mice. The treatment of mice with different doses of PME (400 and 600 mg/kg body weight) for 7 days before the administration of a single i.p. dose of cisplatin (5 mg/kg) exhibited significant chemoprotective activity. Renal dysfunction was evaluated biochemically by measuring the concentration of blood urea nitrogen (BUN) and serum creatinine and histologically by light microscopy. Genotoxicity was evaluated by the bone marrow micronucleus assay. A single dose of cisplatin significantly elevated the levels of blood urea nitrogen, serum creatinine, and the kidney to body weight ratio, but pretreatment with PME (600 mg kg−1 day−1) for 7 days significantly attenuated the cisplatin-induced nephrotoxicity. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow was determined at 24 h after the administration of cisplatin. After administration of cisplatin, the frequency of MNPCEs distinctly increased. In mice treated with PME before cisplatin application, there was a decrease in the number of MNPCEs when compared with mice injected with only cisplatin. Ethanol extract of PME thus has a marked free radical scavenging effect indicating its antioxidative property. The results suggest that the ethanol extract of PME has a protective effect against cisplatin-induced nephro- and genotoxicity through its antioxidant property.

Protective effect of lipoic acid on micronuclei induction by cyclophosphamide

The present study investigated the protective efficacy of DL-alpha-lipoic acid on the cyclophosphamide (CP)-induced clastogenicity using the in vivo micronucleus assay. Male Wistar rats of 140 +/- 20 g were categorized into eight groups. Five groups were administered CP (40 mg/kg body weight, intraperitonealy) to induce genotoxicity; four of these groups received a single intraperitoneal injection of lipoic acid at a dose of either 100 or 200 mg/kg body weight, and either 30 or 60 min prior to CP administration. A vehicle-treated control group and lipoic acid control groups were also included. The number of micronucleated polychromatic erythrocytes (MNPCEs) was determined at 24 h after CP administration. In rats injected with CP, the frequency of MNPCEs in bone marrow and peripheral blood was increased significantly in comparison with the controls, and in rats treated with lipoic acid and CP, the number of MNPCEs was decreased significantly in comparison to those given CP alone. The chemoprotective effect was found to be stronger after the administration of lipoic acid at a dose of 200 mg/kg body weight than 100 mg/kg body weight dosage, indicating the dose-dependent protective effect of lipoic acid. However, the protection by lipoic acid was not dependent on the time intervals between lipoic acid and CP administration. Our results illustrate the protective effect of lipoic acid on the in vivo clastogenicity induced by CP.

In vivo genotoxic evaluation of the furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene in mouse bone marrow

The genotoxic potential of the compound 1-(5-bromofur-2-yl)-2-nitroethene (2-βNF) has been tested by using the in vivo mouse bone marrow micronucleus assay. Its ability to induce clastogenicity or aneugenicity, through the induction of micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow cells has been evaluated. Treatment groups of five CD-1 male mice were administered once intraperitoneally at the doses of 10, 20, and 30 mg/kg, and their bone marrows were sampled at 24 and 48 h after the administration, at the first sampling time animals administered with the three doses were used, and in the second sampling time, only animals administered with the highest dose were used. All animals treated with the highest dose of the test compound (30 mg/kg) showed evident clinical symptoms of toxicity such as irritation, hunched posture, slight ataxia, dyspnoea, piloerection, and palpebral ptosis. However, no marked depression of bone marrow cell proliferation was observed, and no significant increases in the frequency of MNPCE were obtained in any of the concentrations tested at any sampling times. The positive control treated-animals were administered with cyclophosphamide at the dose of 40 mg/mL. The compound caused a significant increase in the number of MNPCE in all treated animals, demonstrating the sensitivity of the mouse strain used. From the results obtained, it is concluded that the compound 2-βNF is neither clastogenic nor aneugenic in the erythrocytes from the bone marrow of treated mice at the doses tested.

Radioprotective effects of the thiols GSH and WR-2721 against X-ray-induction of micronuclei in erythroblasts

The frequency of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow and peripheral blood of adult male Swiss mice treated with reduced glutathione (GSH) and S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721), at a dose of 400 mg/kg body weight, and exposed to 6 Gy X-rays. GSH or WR-2721 was applied alone, or 60 and 30 min, respectively, prior to X-ray-exposure. The number of MNPCEs was determined at 24 h after the thiol treatment and X-irradiation. The radioprotection and toxicity caused in the mouse erythroblasts by GSH and WR-2721, as indicated by the number of MNPCEs were dependent on the thiol applied. The stronger radioprotective effect is obtained following WR-2721 administration than after GSH application. WR-2721 showed greater toxicity than GSH. The combination of GSH and WR-2721 given before X-ray-exposure resulted in the most radioprotective effect as compared to the respective single-drug treatment of mice. Application of the both thiols, without subsequent X-irradiation appeared to be the most toxic, compared with administration of WR-2721 or GSH alone. The effective radioprotection by the combined action of GSH and WR-2721 against genomic instability induced in the mouse erythroblasts by X-rays was shown.

WR-2721: inhibitor of cisplatin-induced micronuclei.

The modulatory effect of S-2-/3-aminopropylamino/ethyl-phosphorothioic acid, (WR-2721, Amifostine) on induction of micronuclei by cis-diamminedichloroplatinum[II] (CDDP) was studied. The adult, male Swiss mice were treated with WR-2721, at a dose of 200 mg/kg or 400 mg/kg body weight, and/or with CDDP, at a dose 5 mg/kg or 10 mg/kg body weight. WR-2721 was given alone or 30 min before CDDP administration. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) and also the number of polychromatic erythrocytes (PCEs) in the bone marrow and peripheral blood, at 24 h after the drug application, were determined. After administration of CDDP, the frequency of MNPCEs distinctly increased, and the number of PCEs decreased. As compared with the animals injected with CDDP only, in mice treated with WR-2721 before CDDP application, the number of MNPCEs was reduced and the frequency of PCEs was increased. However, WR-2721 given alone, without subsequent administration of cis-diamminedichloroplatinum[II], caused an increase in the number of micronucleated polychromatic erythrocytes and a decrease in the number of PCEs. The geno- and cyto-toxicity and chemoprotection were dependent on the doses of the agents WR-2721 and CDDP applied. In mice injected with CDDP and/or WR-2721, the patterns of changes in the frequency of MNPCEs and PCEs were similar in the bone marrow and peripheral blood, respectively. The protective effect of the aminothiol compound WR-2721 against induction of micronuclei and apoptotic cell death in the erythropoietic system by CDDP was shown.

Effects of GSH and WR-2721 on induction of micronuclei by cyclophosphamide

The frequency of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow and peripheral blood of adult male Swiss mice treated with reduced glutathione (GSH) and/or S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721), at a dose of 400 mg/kg body weight, and/or with cyclophosphamide (CP), at a dose of 200 mg/kg body weight. GSH was given 60 or 15 min and/or WR-2721 was applied 30 min before CP administration. The number of MNPCEs was determined at 24 h after the drug application. After treatment of mice with CP, the frequency of MNPCEs was distinctly increased. The stronger chemoprotective effect against CP-induced cytotoxicity was obtained following GSH administration than after WR-2721 injection. WR-2721 characterized greater cytotoxicity than GSH. The combination of GSH and WR-2721 given alone, or before CP administration resulted in the most cytotoxic and chemoprotective effects, compared with the respective single-thiol treatment of mice. The most effective protection against CP-induced genotoxicity was observed in the case of treatment of mice with WR-2721and GSH, respectively, 30 and 15 min before CP administration. The most cytotoxic effect of the thiols was found when GSH given 30 min prior to WR-2721 application. The chemoprotection and cytotoxicity caused in the mouse erythroblasts by GSH and WR-2721, as indicated by the number of MNPCEs were dependent on the thiol(s) given, and the time intervals between the drug administration. The modulatory effect of the thiols GSH and WR-2721 on ‘delayed apoptosis’ induced in the erythropoietic system by cyclophosphamide was shown.

Induction of micronucleated erythrocytes by MEA, AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.

Effects of quinoline and 8‐hydroxyquinoline on mouse bone marrow erythrocytes as measured by the micronucleus assay

Both quinoline and 8-hydroxyquinoline (HOQ) were tested for their genotoxicity in CD1 male mice by using a bone marrow micronucleus assay. Mice were intraperitoneally treated in single injections with three dose levels (25, 50, and 100 mg/kg) of each chemical with corn oil as solvent vehicle. Bone marrow was sampled at 24, 48, and 72 h postinjection. Quinoline resulted in a significant dose-related increase in the number of micronucleated polychromatic erythrocytes (MPCE) at the 24 h sampling time for all doses tested. The high dose (100 mg/kg) and the medium dose (50 mg/kg) also induced statistically significant increases (P less than .05) in the number of MPCEs at 48 h interval. The ratios of polychromatic to normochromatic erythrocytes at the 24 h sampling time were lower for the treated than the control animals. Although HOQ resulted in some increases in the number of MPCEs over the control, this compound induced a statistically significant increase in the number of micronucleated normochromatic erythrocytes (MNCEs) at all three doses following 24 h treatment. Both low and medium doses also induced a higher incidence of MNCEs at the 48 and 72 h sampling times. No data were available for the high dose at these times. The cytotoxic effect of this compound was expressed as low PCE/NCE ratios with all doses at 24 h after injection and as a high mortality rate in animals treated with the high dose (100 mg/kg).