Synergistic immunostimulating activity of pidotimod and red ginseng acidic polysaccharide against cyclophosphamide-induced immunosuppression

We investigated the synergistic effect of combined treatment with red ginseng acidic polysaccharide (RGAP) from Panax ginseng C.A. Meyer and pidotimod in cyclophosphamide-treated mice. The combination of pidotimod and RGAP restored concanavalin A-induced splenic T cell proliferation and LPS-stimulated B cell proliferation significantly. The production of nitric oxide from peritoneal macrophages was increased by the combinations. NK cell activity was increased by RGAP alone or in combination with pidotimod. A synergistic increase in the level of serum IL-12 and interferongamm was observed when the combination of the two was used. RGAP alone or in combination with pidotimod modulated the level of serum C-reactive protein to a near-normal level. These results indicate that combinations of pidotimod and RGAP are synergistic and suggest that combination therapy using pidotimod and RGAP for improving immune activity may provide an additional benefit over the use of the two drugs by themselves.     

肌肽对环磷酰胺毒性影响的初步研究

目的研究肌肽减少环磷酰胺毒性的效果。方法连续15天给小鼠注射环磷酰胺和肌肽，定期检测小鼠的体重，白细胞和红细胞及骨髓细胞的变化情况，并记录60天内小鼠的存活数。15天后取小鼠肝脏，测丙二醛的含量。结果肌肽明显提高了小鼠的白细胞和骨髓细胞的数目，降低了丙二醛的含量，并延长了小鼠的生存期，但对小鼠的体重没有改善作用。结论肌肽对环磷酰胺的毒性有一定的缓解作用。     

Amelioration of cyclophosphamide-induced hepatotoxicity by the root extract of Decalepis hamiltonii in mice

Hepatoprotective potential of the aqueous extract of the roots of Decalepis hamiltonii (DHA) against cyclophosphamide (CP)-induced oxidative stress has been investigated in mice. Administration of CP (25 mg/kg b.w., i.p) for 10 days induced hepatic damage as indicated by the serum marker enzymes aspartate and alanine transaminases (AST, ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). Parallel to these changes CP induced oxidative stress in the liver as evident from the increased lipid peroxidation (LPO), reactive oxygen species (ROS), depletion of glutathione (GSH), and reduced activities of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST). Treatment with DHA (50 and 100 mg/kg b.w., po) mitigated the CP-induced oxidative stress. Moreover, expression of genes for the antioxidant enzymes, were down-regulated by CP treatment which was reversed by DHA. Our study shows the DHA protected the liver from toxicity induced by CP and therefore, it could be serve as a safe medicinal supplement during cyclophosphamide chemotherapy.     

Pirfenidone diminishes cyclophosphamide-induced lung fibrosis in mice

The deposition of excess or abnormal collagen characteristic of pulmonary fibrosis can disrupt gas exchange resulting in severe respiratory impairment. There currently are no effective pharmacologic agents available that inhibit the fibrotic process. Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is an investigational drug that, when administered at 0.5% (w/w) of the diet, decreases both histologic and biochemical evidence of lung fibrosis in hamsters treated intratracheally with bleomycin. The effectiveness of pirfenidone against lung fibrosis initiated by a systemically administered agent was investigated in mice treated intraperitoneally with 200 mg/kg cyclophosphamide (CP). Control and treated animals were fed a diet containing 0.277% (w/w) pirfenidone beginning 1 day after CP. Despite anorexia in the CP-treated mice the first day after treatment, they ingested a greater average pirfenidone dose over 20 days than saline-treated control mice (717±44 versus 564±30 mg/kg per day, respectively). Total lung hydroxyproline content, an index of fibrosis, was significantly lower 21 days after treatment with CP plus pirfenidone as compared to mice treated with CP alone. Although microscopic lung fibrosis scores were not significantly decreased by pirfenidone in CP-treated mice, the overall incidence of fibrosis was significantly decreased. Histologically, mice treated with CP showed fibrosis while mice treated with CP plus pirfenidone exhibited fewer abnormalities. The rate of hydroxyproline synthesis by lung tissue 9 days after treatment with CP was significantly elevated. This rate was not affected by pirfenidone treatment. Overall, these data support an antifibrotic effect of pirfenidone against CP-induced lung fibrosis in mice. The mechanism of its effect is not known, but appears to be unrelated to an inhibition of collagen synthesis.     

Ameliorative effect of propolis against cyclophosphamide-induced toxicity in mice

Abstract

Context: Cyclophosphamide (CTX) is a common anticancer agent used for the treatment of several malignancies. However, upon treatment, it induces severe toxicity due to its oxidative stress capability. Propolis, a natural product collected by honey bees, has shown several biological activities, such as free radical scavenging and antioxidant agent.

Objective: This study elucidates the protective effects of propolis against CTX-induced changes in mice.

Materials and methods: Forty-eight male Swiss albino mice were divided into four groups; group 1 was intraperitoneally (i.p.) injected with 200?µL of phosphate buffer saline (PBS), group 2 was injected with 100?mg/kg/d propolis, group 3 was injected with a single dose of CTX (200?mg/kg), and group 4 was injected with a single dose of CTX (200?mg/kg) followed by propolis (100?mg/kg) for 7 consecutive days. After 12?d, mice were bled and then sacrificed to analyze the hematological, biochemical, and histological parameters.

Results: The results indicated that CTX-injected mice showed an increase in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine and a decrease in the total number of white blood cells (WBCs) and platelets. Moreover, dramatically changes in the histological architectures of the liver and kidney were observed. The mice that were injected with CTX/propolis showed an improvement in the levels of ALT, AST, urea, creatinine, WBCs, and platelets. Moreover, the histological picture of the liver and kidney was significantly improved.

Conclusions: In conclusion, propolis might be considered an effective agent in ameliorating the toxicity resulted from CTX treatment.     

Chemoprotective effects of captopril against cyclophosphamide-induced genotoxicity in mouse bone marrow cells

The protective effects of captopril (CAP) against toxicity induced by cyclophosphamide (CP) in mice were investigated using the micronucleus assay for anticlastogenic activity in mouse bone marrow cells and liver glutathione (GSH) content. A single intraperitoneal (i.p.) injection of CAP at 50, 100, and 200 mg/kg 1 h prior to cyclophosphamide (50 mg/kg) reduced the frequency of micronucleated polychromatic erythrocytes (MnPCEs). All three doses of CAP significantly reduced the frequency of MnPCEs in mouse bone marrow compared to the group treated with CP alone (P<0.0001–0.01). CP significantly depleted the GSH content in liver but the application of CAP at a dose of 100 mg/kg 1 h before CP treatment repleted the GSH content. CAP exhibited concentration-dependent antioxidant activity, scavenging >96% of the 1,1-diphenyl-2-picryl hydrazyl free radicals when used at a concentration of 0.2 mM. It appears that CAP, due to its antioxidant activity and by increasing GSH levels, can modulate the reduced cellular thiol content induced by CP and reduce the genotoxicity of CP in bone marrow cells.     

Contribution of flavonoid antioxidants to the preventive effect of mesna in cyclophosphamide-induced cystitis in rats

Cyclophosphamide (CP) is widely used, alone or in combination with other chemotherapeutic agents, for treatment of neoplastic diseases. Its urotoxicity may cause dose-limiting side-effects, for example hemorrhagic cystitis. The agent most often used to prevent this side-effect is mesna (2-mercaptoethane sulfonate). Overproduction of reactive oxygen species during inflammation is one reason for possible urothelial injury. The aim of this study was to evaluate whether combinations of quercetin and epigallocatechin 3-gallate (EGCG), flavonoid antioxidants and mesna could prevent cystitis induced by cyclophosphamide, better than mesna alone. A total of 38 male Sprague–Dawley rats were divided into five groups. Four groups received single dose of CP (100 mg kg^–1) intraperitoneally at the same time. Group 2 received CP only, group 3 received mesna (3×21.5 mg kg^–1), group 4 received a single dose of mesna+EGCG (2×20 mg kg^–1), and group 5 received a single dose of mesna+quercetin (2×20 mg kg^–1), before and after CP injection. Group 1 (not treated) served as control. CP injection alone resulted in severe cystitis. Mesna resulted in some, but not full, protection against CP toxicity. Quercetin and catechine, together with mesna, resulted in full protection against CP toxicity, on the basis of histopathology of the urinary bladder. It was concluded that oxidants might be important in the pathogenesis of CP-induced cystitis, and that flavonoid antioxidants, used in addition to mesna, may help to ameliorate bladder damage.This revised version was published online in May 2005 with correction to the author Ahmet Korkmaz     

Effects of cyclophosphamide on the phenotypes and functions of THP-1 cells

Early and accurate evaluation of immunotoxicity is crucial. However, there are few in vitro models for immunosuppressive evaluation. THP-1 cells has long been used for in vitro sensitivity evaluation. Whether it can be used for immunosuppressive evaluation remains unclear. In this study, effects of immunosuppressant cyclophosphamide (CY) on THP-1 cells were observed while 2, 4-Dinitrochlorobenzene (DNCB) was used as a control. The phenotypes of THP-1 cells, the ability to activate naïve T cells, intracellular reactive oxygen species (ROS) level, gene markers, phagocytic ability and cell apoptosis were detected after THP-1 cells being exposed to different concentrations of CY and DNCB. Both CY and DNCB were able to activate THP-1 cells, but there were a lot of differences in their effects on THP-1 cells, such as the changes in phenotypes, in the ability to activate naïve T cells, in ROS production and in marker gene expression. Firstly, CY down-regulated the expression of CD86 on THP-1 cells while DNCB up-regulated its expression. Secondly, the ability of THP-1 cells to activate naïve T cells was enhanced by CY and suppressed by DNCB. Thirdly, CY raised rapid and transient elevation of ROS level in THP-1 cells, while the effects of DNCB were slower and longer-lasting. Finally, only CY could lead to an increase in heme oxygenase 1 (HMOX1) expression. Taken all these results into account, we suggested that THP-1 cell line possesses the potency to be an in vitro model of immunosuppressive evaluation. And the surface molecule CD86, the ability to activate naïve T cells, the ROS production and the gene marker HMOX1 of THP-1 cells are promising markers.     

复方HD对环磷酰胺致免疫抑制小鼠免疫功能的影响

目的 探讨复方HD对环磷酰胺诱导的免疫抑制小鼠的免疫增强作用。方法 ICR小鼠分别以蒸馏水（对照组、模型组）、香菇多糖（200 mg/kg）、复方HD（5、10、20 g/kg）每天1次ig给药,连续10 d;除对照组外,在第4~6天ip环磷酰胺（40 mg/kg）制备免疫抑制小鼠模型（对照组除外）;每天给药前精确称量并记录小鼠体质量。于最后一次给药24 h后,测定小鼠脾指数、胸腺指数;流式细胞术进行全血中T细胞亚群分析;ELISA法检测血浆中IFN-γ和IL-4水平。结果 与模型组比较,复方HD低、中、高剂量组显著提高小鼠的胸腺指数,CD3⁺、CD3⁺CD4⁺细胞数以及CD4⁺/CD8⁺比值（P<0.05）,显著恢复血浆中IFN-γ水平和IFN-γ/IL-4比值（P<0.05）,纠正了Th1/Th2偏移,并呈现出一定的浓度相关性。结论 复方HD能够促进环磷酰胺诱导的免疫抑制小鼠免疫功能的恢复,证明其在预防及治疗免疫功能低下方面具有一定的疗效。     

Effect of N-acetylcysteine on some aspects of cyclophosphamide-induced toxicity and immunosuppression.

Cyclophosphamide (CPA)-induced bladder toxicity and lethality were inhibited by N-acetylcysteine (NAC) when given systemically at a 4 : 1 ratio prior to CPA. This dose of NAC did not affect the production or the formation of free alkylating agents from CPA in vivo or in vitro. The immunosuppressive effect of CPA against T cell response systems, such as graft vs host reaction, antibody to sheep red blood cells and (PHA) phytohemagglutin stimulation was unaffected by NAC. It is concluded that the metabolic products of CPA for cytotoxicity as expressed as cystitis and lethality are different from the alkylating agents, which appear to affect immunological phenomena.     

Pretreatment with 1,8-cineole potentiates thioacetamide-lnduced hepatotoxicity and immunosuppression

The effect of 1,8-cineole on cytochrome P450 (CYP) expression was investigated in male Sprague Dawley rats and female BALB/c mice. When rats were treated orally with 200, 400 and 800 mg/kg of 1,8-cineole for 3 consecutive days, the liver microsomal activities of benzyloxyresorufin- and pentoxyresorufin-omicron-dealkylases and erythromycin N-demethylase were dose-dependently induced. The Western immunoblotting analyses clearly indicated the induction of CYP 2B1/2 and CYP 3A1/2 proteins by 1,8-cineole. At the doses employed, 1,8-cineole did not cause toxicity, including hepatotoxicity. Subsequently, 1,8-cineole was applied to study the role of metabolic activation in thioacetamide-induced hepatotoxicity and/or immunotoxicity in animal models. To investigate a possible role of metabolic activation by CYP enzymes in thioacetamide-induced hepatotoxicity, rats were pre-treated with 800 mg/kg of 1 ,8-cineole for 3 days, followed by a single intraperitoneal treatment with 50 and 100 mg/kg of thioacetamide in saline. 24 h later, thioacetamide-induced hepatotoxicity was significantly potentiated by the pretreatment with 1,8-cineole. When female BALB/c mice were pretreated with 800 mg/kg of 1,8-cineole for 3 days, followed by a single intraperitoneal treatment with 100 mg/kg of thioacetamide, the antibody response to sheep red blood cells was significantly potentiated. In addition, the liver microsomal activities of CYP 2B enzymes were significantly induced by 1,8-cineole as in rats. Taken together, our results indicated that 1,8-cineole might be a useful CYP modulator in investigating the possible role of metabolic activation in chemical-induced hepatotoxicity and immunotoxicity.     

Effects of ascorbic acid on the mouse embryo and on cyclophosphamide-induced cephalic DNA strand breaks in vivo

Pregnant C₃H mice were exposed to 3.34 and 6.68 g ascorbic acid/kg body weight on the 11th day postcopulation, and to co-administration of a teratogenic dose of cyclophosphamide (CP, 15 mg/kg body weight). The effects on embryonal cephalic DNA strand breaks were assessed 16 h after drug administration. In order to establish whether vitamin C was embryotoxic or altered CP-induced toxicity, mice were sacrificed on day 18 after copulation to record fetal weights, gross morphological abnormalities, and fetal mortality. Administration of 3.34 g ascorbate/kg was not associated with demonstrable toxic effects but with 6.68 g ascorbic acid/kg there was a 46% incidence of fetal mortality. In embryos exposed to CP, 15 mg/kg, there was a decrease in fetal weight (median fetal weight 678 mg compared with 967 mg in controls), all fetuses were morphologically abnormal and 59% of cephalic DNA was double stranded compared with 81% for controls (p<0.001). When vitamin C, 3.34 g/kg, was co-administered with CP the incidence of DNA strand breaks remained unchanged. However, all fetuses were morphologically normal and there was no reduction in fetal weight. These findings demonstrate that administration of 6.68 g vitamin C/kg is toxic to the mouse embryo, but a lower dose of 3.34 g/kg is not, and has a protective effect against the toxic manifestations of CP. This protection is not associated with prevention of cephalic DNA strand breaks.     

Chemoprotective effect of lipoic acid against cyclophosphamide-induced changes in the rat sperm

Treatment with cyclophosphamide (CP), a commonly used anticancer and immunosuppressive agent, may result in oligospermia and azoospermia. CP administration induces oxidative stress and is cytotoxic to normal cells. In this context, we have studied the effect of an established antioxidant, lipoic acid on its influence on CP-induced oxidative injury in rat sperm. In this study, we have assessed the possible protective efficacy of lipoic acid on the sperm characteristics, peroxidative damages and abnormal antioxidant levels in the epididymal sperm of CP-administered rats. Male Wistar rats of 140+/-20 g were categorized into four groups. Two groups of rats were administered CP (15 mg/kg body weight once a week for 10 weeks by oral gavage) to induce testicular toxicity; one of these groups received lipoic acid treatment (35 mg/kg body weight intraperitoneally once a week for 10 weeks; 24 h prior to CP administration). A vehicle treated control group and a lipoic acid drug control group were also included. CP-treated rats showed a significant decrease in sperm count and motility with an increase in dead and abnormal sperms. The epididymal sperm of untreated CP-exposed rats showed 1.9-fold increase in lipid peroxidation, along with a significant increase in protein carbonyl level. These changes were associated with significant increase in DNA damage in the sperm as evidenced by increased single strand breaks in fluorimetric analysis of DNA unwinding (FADU). In rats treated with CP, abnormal changes in the activities/levels of enzymic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymic (reduced glutathione, ascorbate and alpha-tocopherol) antioxidants, were also observed. Pretreatment with lipoic acid improved the semen quality and reduced the oxidative stress and DNA damage induced by CP, thereby demonstrating the protection rendered by lipoic acid.     

Preventive and therapeutic effects of sugar cane extract on cyclophosphamide-induced immunosuppression in chickens

Effects of oral administration of sugar cane extract (SCE) on immunosuppression in chickens treated with cyclophosphamide (CPA) were evaluated. Three-week-old inbred chickens were inoculated into the crop with SCE (500 mg/kg/day) for three consecutive days before or after injection of CPA 12 or 20 mg/chicken. At the last day of SCE or CPA treatment, all chickens were immunized intravenously with sheep red blood cells (SRBC) and Brucella abortus (BA). Chickens administered SCE showed a significant increase in body weight, gain in body weight/day, relative weight of the bursa of Fabricius and antibody responses to SRBC and BA than untreated control chickens. Chickens injected with CPA alone showed significantly decreased body weight, gain in body weight/day, relative weight of the bursa and antibody responses to SRBC and BA, showing immunosuppression in the bursa-dependent immune system. All chickens administered SCE before or after the treatment with CPA showed significantly higher values in body weight, gain in body weight/day, relative bursal weight and antibody responses to both antigens, when compared to chickens treated with CPA alone. In histological examination, chickens administered SCE showed a typical bursa with well constituted follicles, although chickens treated with CPA alone showed a severely atrophied bursa with rudimentary follicles and enormous proliferation of interfollicular connective tissue. Chickens treated with SCE and CPA showed a well-reconstituted bursa with almost normal structure. These results suggest that SCE has functionally and morphologically reconstituting effects on the bursa-dependent immune system in immunosuppressed chickens induced by injection of CPA.     

叔丁基对苯二酚上调Nrf2调控的基因表达并减轻环磷酰胺导致的小鼠血液毒性

血液毒性（或称骨髓抑制）是癌症化疗最常见的剂量限制毒副作用,而转录因子Nrf2控制着包括骨髓在内的许多组织对化学刺激的敏感性。本文研究了叔丁基对苯二酚（tBHQ）对小鼠外周血细胞中Nrf2调控的基因表达和环磷酰胺（CTX）导致的血液毒性的影响。CTX处理导致了小鼠外周血有核细胞的凋亡和白细胞减少,伴随着骨髓造血细胞的动员。tBHQ处理则可以在体外和体内激活RAW264.7小鼠巨噬细胞和外周血细胞中Nrf2信号和下游基因如血红素氧化酶1和谷胺酸半胱氨酸连接酶催化亚基等的表达同时,tBHQ预处理可以减轻CTX导致的小鼠外周血有核细胞的凋亡和白细胞减少,说明Nrf2可能在减轻CTX血液毒性中发挥作用。本文的研究有助于加深对化疗所致血液毒性的了解,并提示Nrf2可能作为减轻化疗毒副作用的化疗保护剂的药物靶标。     

Acute infection of mice with highly virulent group B streptococci as a host resistance model for immunotoxicity assessment

This report describes a unique model for immunotoxicity evaluation in mice. The model is adapted from previously described mouse models for group B streptococcus (GBS) infections in human neonates. In this disease as well as a number of human diseases caused by highly virulent pathogens, the mechanisms of innate immunity are unable to protect the host, and survival is strictly dependent on acquired immunity. Unlike other host resistance models widely used in immunotoxicity studies, the GBS model utilizes bacteria that are highly virulent for mice (LD₅₀=5–17 colony forming units). GBS is not virulent for adult humans and can be safely handled with typical precautions. Acquired immunity in the GBS model is induced during a 2 week period by two injections of heat-killed GBS. The immunizing doses are the minimum which will allow survival of 80–100% of mice in response to challenge with an otherwise lethal dose of live GBS (100 bacteria). Administration of the immunotoxic agents cyclophosphamide, carrageenan, or cobra venom factor during the immunization period and/or shortly before challenge significantly suppressed host resistance. For example, the composite mortality rate for unimmunized mice was 98% and the rate for immunized mice was 8.5%. For all groups treated with cyclophosphamide (one 75 mg/kg dose 48 h before each immunization) the mean mortality was 41 ± 18%. The consistency of the model was evaluated by repetition of several treatments in independent experiments, and the model's consistency is comparable to that of other host resistance models. Finally, bacterial concentrations in the blood or peritoneum 16 h after challenge were evaluated as possible endpoints for the GBS model, and GBS concentration in the peritoneum seems to be a reliable indicator of immunotoxicity. Measurement of GBS concentration was comparable to survival time in discerning statistically significant differences between groups, and both of these parameters were superior to per cent mortality, particularly when small groups (six mice per group) were used.     

Effects of a 5-HT1A receptor agonist on acute and delayed cyclophosphamide-induced vomiting

LY228729 [(−)-4(dipropylamino)-1,3,4,5-tetrahydrobenz-{c,d}indole-6-carboxamide}], an agonist at the 5-HT_1A subtype of 5-HT receptor, was studied as an antiemetic in pigeons dosed with a highly emetic oncolytic agent, cyclophosphamide. An intramuscular injection of 0.32 mg/kg of LY228729 administered 15 min prior to the intravenous injection of 200 mg/kg of cyclophosphamide totally prevented the acute emetic response induced by cyclophosphamide. When used as a rescue therapy in a separate group of pigeons, LY228729 (0.32 mg/kg, i.m.) prevented further emetic episodes when it was administered after vomiting had already been induced by cyclophosphamide. Injections of LY228729 given at intervals over the next 2 d also attenuated the delayed emetic response induced by cyclophosphamide. LY228729 appears to be a broad spectrum antiemetic agent that is effective against the anticipatory, the acute and the delayed stages of emesis induced by oncolytic agents.     

Attenuation of cyclophosphamide-induced taste aversions in mice by prochlorperazine, delta 9-tetrahydrocannabinol, nabilone and levonantradol

A series of experiments were performed with adult CD-1 male mice to evaluate the antiemetic effects of several compounds using the conditioned taste aversion procedure. The antiemetics were administered IP immediately prior to a 30-min conditioning trial in which a novel tasting solution (0.3% saccharin) was presented to the subjects. The emetics, apomorphine and the cancer chemotherapeutic drug cyclophosphamide, were given IP immediately after the conditioning trial at doses that induced taste aversions. Three days later the mice received a two bottle preference test (saccharin vs. water) and the percent saccharin consumed of the total fluid intake was calculated. Doses of the phenothiazine antiemetic prochlorperazine (1 and 3 mg/kg) attenuated the aversions produced by 0.3 and 1.0 mg/kg apomorphine. Doses of drugs currently approved or under clinical investigation as antiemetics in conjunction with cancer chemotherapy, i.e., prochlorperazine (1.0 mg/kg), delta 9-tetrahydrocannabinol (0.3 and 1.0 mg/kg) and nabilone (0.01 and 0.03 mg/kg), significantly attenuated the taste aversions induced by cyclophosphamide. Levonantradol at doses of 0.03 and 0.06 mg/kg, however, did not attenuate cyclophosphamide-induced taste aversions. Conditioned taste aversions produced by emetic drugs warrants investigation as a model for evaluating potential antiemetics.