Influence of teniposide (VM-26) on radiation-induced damage to mouse spermatogenesis: a flow cytometric evaluation

The effect of teniposide (VM-26) 0.05 mg/kg body weight treatment on spermatogenesis of mice exposed to 0, 0.5, 1, 2, and 3 Gy gamma-radiation was evaluated flow cytometrically. Whole body irradiation with 1 to 3 Gy resulted in a significant decline in testis weight from Day 14 to 35 post-irradiation depending on the exposure dose. Treatment of mice with teniposide before irradiation advanced the decline in testicular weight by several days, especially at 3 Gy, where a significant decline in testicular weight was observed at Day 7 post-irradiation when compared with the double distilled water (DDW)+irradiation group. The relative percentage of the 2C population declined significantly in the VM-26+irradiation group in comparison with the DDW+irradiation group at various post-irradiation time periods depending on the exposure dose. A significant depletion in the relative percentage of S-phase cells was observed as early as Day 1 post-irradiation in the VM-26+irradiation group when compared with the DDW+irradiation group after exposure to 1 to 3 Gy. This decline continued up to Day 21 post-irradiation after exposure to 2 Gy. The relative percentage of primary spermatocytes showed a consistent decline after exposure to various doses of gamma-radiation in the VM-26+irradiation group when compared with the DDW+irradiation group at different time periods, with a few exceptions, especially at higher doses. The pattern of decline in the relative percentage of round spermatids was similar to that of primary spermatocytes, where a significant decline was observed at various post-irradiation time periods in the VM-26+irradiation group in comparison with the DDW+irradiation group. These changes in the relative germ cell percentages are manifested as alterations in the ratios of various germ cell populations. The 4C:2C ratio declined consistently from Day 1 to Day 70 post-irradiation in the VM-26+irradiation group at all exposure doses. Similarly, the 4C:S-phase ratio in the VM-26+irradiation group also showed a significant decline at different post-irradiation time periods when compared with the DDW+irradiation group depending on the exposure dose. The reduction observed in the relative percentages of various cell populations was higher in the combination group when compared with the DDW+irradiation controls, indicating potentiation of damage to male germ cells by teniposide treatment before irradiation.     

Elevation of micronuclei frequency in mouse bone marrow treated with various doses of teniposide (VM-26)

The effect of various doses (0-10 mg/kg body wt.) of teniposide (VM-26) was studied on the induction of micronuclei at 12, 24 and 36 h post-treatment. The frequency of micronuclei (MPCE and MNCE) increased in a dose-dependent manner up to a dose of 0.3125 mg/kg VM-26, where a peak frequency of micronuclei was observed. A further increase in the drug dose resulted in the reduction in micronuclei frequency in comparison with 0.3125 mg/kg drug dose reaching a nadir at 10 mg/kg. However, it was significantly higher than DDW (double distilled water) treated controls. The pattern of micronuclei induction was similar for all the post-treatment time periods. The frequency of micronuclei also increased with scoring time and the highest frequency of micronuclei was observed at 24 h post-treatment, which declined thereafter without restoration to DDW treated control level. Conversely, the PCE/NCE ratio registered a dose-dependent decline after treatment of mice with various doses of VM-26. A peak decline was observed at a dose of 0.3125 mg/kg, thereafter the decline became consistently less resulting in an elevation in the PCE/NCE ratio in comparison with 0.3125 mg/kg VM-26.     

Flow cytometric evaluation of the effect of various doses of vindesine sulphate on mouse spermatogenesis

Spermatogenesis, a rapidly proliferating cell system, is highly susceptible to damage by radiotherapy and/or chemotherapy. Vindesine, a semisynthetic vinca alkaloid, was given as a single injection to adult male Swiss albino mice to study its effects on testicular weight and male germ cell turnover pattern using flow cytometry. Testicular weight declined significantly at Day 7 to 14 and from Day 14 to 35 after administration of 1 and 2 mg/kg b wt vindesine, respectively. Flow cytometric evaluation of various testicular cell types after the administration of 2 mg/kg b wt vindesine revealed a significant increase in the relative percentage of spermatogonial cells at Day 21 and 35 posttreatment. In contrast, the relative percentage of primary spermatocytes declined significantly at Day 7 and 14 posttreatment. Similarly, a significant reduction in the relative percentage of round spermatids was observed from Day 7 to 35 posttreatment. The relative percentage of elongated spermatids declined significantly at day 35 post-treatment. These changes are reflected in the transformation ratios. While the 4C:2C ratio did not exhibit any significant change below 1 mg/kg vindesine, it declined significantly after 1 mg/kg (Day 14) and 2 mg/kg (Day 7 to 35, except Day 28 posttreatment) vindesine treatment. Treatment of male mice with 2 mg/kg vindesine resulted in a significant decline in 1C:2C ratio from 7 to 35 d post-treatment. The 4C:S-phase ratio decreased significantly at Day 7 and 14 posttreatment for all the drug doses above 0.05 mg/kg. A significant reduction in the 1C:4C ratio was observed at day 21 to 35 posttreatment as a result of 2 mg/kg vindesine administration.     

Effect of teniposide (VM-26) on the cell survival, micronuclei-induction and lactate dehydrogenase activity on V79 cells

The genotoxic effect of 0, 1, 5, 10, 25, 50 and 100 nM teniposide (VM-26) treatment was studied on cultured V79 cells. Treatment of V79 cells with different concentrations of teniposide resulted in a concentration-dependent decline in the cell survival and growth kinetics. VM-26 treatment also caused alteration in the cell proliferation kinetics as evidenced by the increase in the frequency of mononucleate cells, with a consequent decline in the frequency of binucleate cells in a concentration-dependent manner at all the post-treatment time periods. Exposure of V79 cells to different concentrations of VM-26 resulted in a concentration related elevation in the frequency of micronucleated binucleate (MN) cells. The frequency of MN was significantly higher in VM-26 treated cells than that of non-drug treated cells at all the post-treatment time periods. A peak frequency of MN was observed at 16 h post-treatment that declined thereafter. The release of lactate dehydrogenase increased with the increase in drug concentration and a maximum LDH release was observed at 0.5 h post-treatment after exposure to 10-100 nM VM-26. While a peak value was observed at 1 and 2 h for 5 and 1 nM VM-26, respectively. The biological response was evaluated by determining the relationship between micronuclei and cell survival. The cell survival declined with increasing MN frequency, resulting in a close but an inverse relationship between the cell survival and micronuclei-induction. The dose effect relationships for micronuclei induction, LDH release and biological response was linear quadratic.     

Correlation between cell survival, micronuclei-induction, and LDH activity in V79 cells treated with teniposide (VM-26) before exposure to different doses of gamma radiation.

The influence of teniposide (VM-26) treatment was studied on the radiation-induced alterations in cell survival, micronuclei (MN) formation and lactate dehydrogenase (LDH) release in V79 cells. Treatment of V79 cells with 10 nM teniposide before exposure to different doses of gamma radiation resulted in a significant decline in the cell survival when compared with the PBS + irradiation group. The decline in cell survival was dose related. The cell proliferation indices also declined in a dose-dependent manner in both PBS + irradiation and VM-26 + irradiation groups. The decline was higher in the VM-26 + irradiation group in comparison with the PBS + irradiation group. In contrast, the frequency of micronuclei increased in a dose-related manner in both PBS + irradiation and VM-26 + irradiation groups. However, the frequency of micronuclei was significantly greater in the latter group when compared with the former group at all the post-irradiation time periods studied. The LDH contents increased in a dose-dependent manner in both PBS + irradiation and VM-26 + irradiation groups at all the post-irradiation time periods evaluated. This elevation in LDH contents was significantly greater in the VM-26 + irradiation group in comparison with the PBS + irradiation group.     

苯妥英钠对化学治疗耐药胶质瘤细胞内卡莫司汀和替尼泊苷药物浓度的影响

目的 研究苯妥英钠对化学治疗耐药人胶质母细胞瘤细胞(8-MG-BA)内卡莫司汀、替尼泊苷积聚浓度的影响.方法 实验细胞分为5组,H4细胞组、8-MG-BA细胞组、8-MG-BA+苯妥英钠5 mg,/L组、8-MG-BA+苯妥英钠10 mg/L组、8-MG-BA+维拉帕米5 mg/L组.采用高效液相色谱法(HPLC)检测各组细胞内卡莫司汀、替尼泊苷积聚浓度.采用外标标准曲线法,以药物的质量浓度(C)为横坐标、峰面积(A)为纵坐标进行线性回归计算,并测定仪器精密度与方法回收率.结果 吸收1、3h后,H4细胞组细胞内卡莫司汀、替尼泊苷含量均高于8-MG-BA细胞组[吸收1h后卡莫司汀浓度:(1.75±0.05)mg/L比(0.31±0.03)mg/L,吸收3h后卡莫司汀浓度:(1.70±0.03)mg/L比(0.39±0.04)mg/L,吸收1h后替尼泊苷浓度:(1.18±0.03)mg/L比(0.42±0.03)mg/L,吸收3h后替尼泊苷浓度:(1.09±0.04)mg/L比(0.46±0.03)mg/L,均P＜0.01];8-MG-BA+维拉帕米5 mg/L组、8-MG-BA+苯妥英钠5 mg/L组、8-MG-BA+苯妥英钠10 mg/L组细胞内卡莫司汀、替尼泊苷含量均高于8-MG-BA细胞组[吸收1h后卡莫司汀浓度:(0.56±0.04)mg/L、(1.10±0.12)mg/L、(1.37±0.04)mg/L比(0.31±0.03)mg/L,吸收3h后卡莫司汀浓度:(0.68±0.04)mg/L、(1.25±0.03)mg/L,(1.49±0.04)mg/L比(0.39±0.04)mg/L,吸收1h后替尼泊苷浓度:(0.50±0.03)mg/L、(0.59±0.03)mg/L、(0.95±0.04)mg/L比(0.42±0.03)mg/L,吸收3h后替尼泊苷浓度:(0.53±0.04)mg/L、(0.62±0.04)mg/L、(1.01±0.03)mg/L比(0.46±0.03)mg/L,均p＜0.01];8-MG-BA+苯妥英钠5 mg/L组、8-MG-BA+苯妥英钠10 mg/L组细胞内卡莫司汀、替尼泊苷含量均高于8-MG-BA+维拉帕米5 mg/L组(P＜0.01);8-MG-BA+苯妥英钠5 mg/L组细胞内卡莫司汀、替尼泊苷含量均低于8-MG-BA+苯妥英钠10 mg/L组(P＜0.05).当被测VM26及卡莫司汀质量浓度为0.05～5 mg/L时,其浓度与峰面积之间具有良好的线性关系.替尼泊苷回收率分别为(96±5)％、(100±4)％和(99±2)％;卡莫司汀回收率分别为(100±5)％、(99±4)％和(99±4)％,日内(24h)、日间(2d间)误差相对标准偏差分别为4.47％和4.96％(n=5).结论 苯妥英钠可以增加化学治疗耐药8-MG-BA细胞内卡莫司汀、替尼泊苷的积聚浓度,并与剂量有关.     

VM-26 in gastric cancer

Summary The Southwest Oncology Group conducted a trial of VM-26 (teniposide) in patients with advanced gastric cancer. VM-26 60 mg/m² IV infusion over 30–45 minutes was given daily for 5 days every 21 days. Twentyone eligible patients with measurable disease and a SWOG performance status of 0–2 were analyzed for response and toxicity. Partial responses were seen in 2 of the 21 eligible patients (9.5%). Median survival was 3.8 months. Severe or life-threatening toxicity was observed in 13/21 (62%) patients. This included two drug related deaths related to neutropenic sepsis and seven other patients with grade 4 granulocytopenia (< 500/mm³). Liver dysfunction and hypotension were seen less often and were not dose limiting. Although the modest activity seen was comparable to that of VP-16 (etoposide) as a single agent, the hematologic toxicity observed in this trial would likely preclude further trials of VM-26 (teniposide) in advanced gastric cancer.     

Induced Cytolethality and Regenerative Cell Proliferation in the Livers and Kidneys of Male B6C3F1 Mice Given Chloroform by Gavage

It has been reported that chloroform administered to male B6C3F₁mice at doses of 138 and 277 mg/kg/day in corn oil by gavage5 days/week for 2 years resulted in incidences of hepato cellularcarcinomas of 36 and 98% relative to an incidence in controlsof 6%. Cytotoxicity and regenerative cell proliferation havebeen implicated in the tumorigenic process for this nongenotoxiccompound. Although chloroform is known to be nephrotoxic inthe male mouse, no treatment-related increase was observed inthe frequency of kidney tumors. To better understand the relationshipof these endpoints, this study evaluated chloroform-inducedcytotoxicity and cell proliferation in the liver and kidneyunder conditions of the cancer study. B6C3F₁ mice were administeredoral doses of 0, 34, 90, 138, or 277 mg/kg/day of chloroformdissolved in corn oil for 4 days or 5 days/week for 3 weeks.Bromo-2'-deoxyuridine (BrdU) was administered via osmotic pumpsimplanted 3.5 days prior to necropsy to label cells in S-phase.Cell proliferation was evaluated in tissue sections immunohistochemicallyas the percentage of cells in S-phase (nuclear labeling index;LI). Mice given 34 and 90 mg/kg/day by gavage had mild degenerativechanges in centrilobular hepatocytes after 4 days of treatment,which was absent at 3 weeks. Centrilobular necrosis was observedin mice given 138 or 277 mg/kg chloroform for 4 days, with increasedseverity of necrosis at 3 weeks. The LI in the livers of micewas increased in a dose-dependent manner at all chloroform dosesfollowing 4 days of treatment. The hepatic LI was similarlyelevated at 3 weeks in the 277-mg/kg dose group, but had declinedin the lower dose groups, and was no longer elevated above controlsat 34 or 90 mg/kg/day. Kidneys from mice in all dose groupsexhibited a dose-related acute tubular necrosis after 4 daysof dosing. After 3 weeks of dosing, regenerating tubules wereobserved in the lower dose groups, while mice treated with 277mg/kg had severe nephropathy characterized by degeneration,necrosis, and regeneration affecting all of the proximal tubules.The LI in the proximal tubules was increased at all doses after4 days of treatment. At the end of 3 weeks of dosing, the LIin the renal cortex had decreased at all dose levels and wasno longer significantly different from controls in the 34- and90-mg/kg/day groups. The sustained increase in LI seen in thelivers of mice administered not only the maximum tolerated dose(MTD), but also one-half the MTD is consistent with the hypothesisthat events secondary to cytolethality and regenerative cellproliferation play a key role in chloroform-induced mouse livercancer. The observation of toxicity and acute regenerative cellproliferation in the kidney without subsequent tumor formationmay be because the severity of the nephrotoxicity in some wayactually interfered with tumor formation. Alternatively, themouse liver may simply be a more sensitive target organ thanthe kidney to tumor formation induced through mechanisms secondaryto regenerative cell proliferation.     

The hepatic endothelial carcinogen riddelliine induces endothelial apoptosis,mitosis, S phase,and p53 and hepatocytic vascular endothelial growth factor expression after short-term exposure

Riddelliineis a naturally occurring pyrrolizidine alkaloid found in certain poisonous rangeland plants of the western United States. In National Toxicology Program 2-year studies, riddelliine induced high incidences of hemangiosarcoma in the liver of F344/N rats (both sexes) and B6C3F1 mice (males). To understand this pathogenesis, we tested short-term effects of riddelliine. Three groups (control; 1.0 mg/kg/day, high dose used in the 2-year study; and 2.5 mg/kg/day) of seven male F344/N rats per group were terminated after 8 consecutive doses and 30 doses (6 weeks, excluding weekends). Serum vascular endothelial growth factor (VEGF), histological, immunohistochemical [factor VIII-related antigen/von Willebrand factor (fVIII-ra/vWf)], VEGF, VEGF receptor-2 (VEGFR2), glutathione S-transferase-pi, S-phase (BrdU), p53, apoptosis, and ultrastructural evaluations were performed on the liver. Following 8 doses of 1.0 and 2.5 mg/kg/day, increased numbers of apoptotic and S-phase nuclei appeared in hepatocytes and endothelial cells. Following 30 doses of 1.0 and 2.5 mg/kg/day, hepatocytes exhibited reduced mitosis, fewer S-phase nuclei, increased hypertrophy, and fatty degeneration, while endothelial cells showed karyomegaly, cytomegaly, decreased apoptosis, more S-phase nuclei, and p53 positivity. Hepatocytes of treated animals expressed higher VEGF immunopositivity. That altered endothelial cells were fVIII-ra/vWf and VEGFR2 positive confirmed their identity. These changes may have promoted hemangiosarcoma development upon long-term exposure through endothelial adduct formation, apoptosis, proliferation of endothelial cells having undamaged and/or damaged DNA, and mutation. Endothelial proliferation may also have been promoted through endothelial arrest at S phase, which was associated with endothelial karyo- and cytomegaly, resulting in hepatocytic hypoxia, triggering VEGF induction.     

Role of vitamin C on lead acetate induced spermatogenesis in swiss mice

In the present study significantly increased lipid peroxidation value (LPP) after a single intraperitioneal injection of lead acetate (LA) (100 mg/kg b.w.) indicated enormous generation of Reactive Oxygen Species (ROS). Lead-induced ROS has a direct inhibitory effect on the growth and differentiation of the spermatogonial cells showing a significant decline in sperm count. Chromosomal analysis of the primary spermatocytes at week 4 post-treatment in lead-treated mice revealed significantly higher no of aberrant cells including chromosomal deficiency, autosomal and XY-asynapsis plates compared to untreated control mice, Sperm morphology studies at week 1-4 and at week 8 post-treatment, indicated higher percentage of deformed sperm population compared to vehicle injected groups of mice. Supplementation of vitamin C (Vit C) at a dose of 10 mg/kg body weight to lead-treated mice groups, however, significantly reduced the LPP with a concomitant increase in sperm count, marked decrease in the no of aberrant cells and significant decline in the percentage of morphologically abnormal sperm population. Protective role of Vit C in combating lead-induced oxidative stress in mice testicular cells, has been discussed.     

Differential up-regulation of striatal dopamine transporter and alpha-synuclein by the pyrethroid insecticide permethrin

The effects of permethrin on striatal dopaminergic biomarkers were assessed in this study. Retired breeder male C57 B1/6 mice were given an ip dose of permethrin (0.1-200 mg/kg) at 7-day intervals, over a 2-week period (Days 0, 7, and 14). Animals were then sacrificed 1 day (t = 1), 14 days (t = 14), or 28 days after the last treatment (t = 28). Dopamine transporter (DAT) protein as assayed by Western blotting was increased to 115% in the 0.8 mg/kg group over that of control mice at t = 1 (P < 0.05). At t = 14, this value increased to 140% of control, and declined slightly to 133% of control at t = 28. The mice given the 1.5 mg/kg dose displayed a significant increase in DAT protein only at t = 28, to 145% of controls. Thus, upregulation of the DAT at low doses of PM is variable 24 h after treatment, and seems to stabilize by t = 28. The threshold dose for increasing DAT expression in Western blots by t = 28 was 0.2 mg/kg permethrin. [(3)H]GBR 12935, used to assay DAT binding, followed the same trend as that for the Western blotting data for 0.8 and 1.5 mg/kg doses of permethrin over the 4 weeks posttreatment. At 200 mg/kg permethrin, DAT protein was unchanged vs controls (t = 1), but had significantly increased by t = 14 and continued to increase at t = 28, suggesting that the reduced dopamine transport at this dose was due to nerve terminal stress and that recovery had occurred. The protein alpha-synuclein was also significantly induced at the 1.5 mg/kg dose at t = 1; however, unlike DAT up-regulation, this effect had declined to control values by t = 14. Maximal induction of alpha-synuclein protein occurred at a dose of 50 mg/kg permethrin. These data provide evidence that the pyrethroid class of insecticides can modulate the dopaminergic system at low doses, in a persistent manner, which may render neurons more vulnerable to toxicant injury.     

Combinations of favipiravir and peramivir for the treatment of pandemic influenza A/California/04/2009 (H1N1) virus infections in mice

Favipiravir, an influenza virus RNA polymerase inhibitor, and peramivir, an influenza virus neuraminidase inhibitor, were evaluated alone and in combination against pandemic influenza A/California/04/2009 (H1N1) virus infections in mice. Infected mice were treated twice daily for 5 d starting 4 h after virus challenge. Favipiravir was 40%, 70%, and 100% protective at 20, 40, and 100 mg/kg/d. Peramivir was 30% protective at 0.5 mg/kg/d, but ineffective at lower doses when used as monotherapy. Combinations of favipiravir and peramivir increased the numbers of survivors by 10-50% when the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir were combined with 20 mg/kg/d favipiravir and when all doses of peramivir were combined with 40 mg/kg/d favipiravir. Three-dimensional analysis of drug interactions using the MacSynergy method indicates strong synergy for these drug combinations. In addition, an increase in lifespan for groups of mice treated with drug combinations, compared to the most effective monotherapy group, was observed for the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir combined with favipiravir at the 20 mg dose level. Therefore, the 20 mg/kg/d dose of favipiravir was selected for further combination studies. Increased survival was exhibited when this dose was combined with peramivir doses of 0.1, 0.25 and 0.5 mg/kg/d (1 mg/kg/d of peramivir alone was 100% protective in this experiment). Improved body weight relative to either compound alone was evident using 0.25, 0.5, and 1 mg/kg/d of peramivir. Significant reductions in lung hemorrhage score and lung weight were evident on day 6 post-infection. In addition, virus titers were reduced significantly on day 4 post-infection by combination therapy containing favipiravir combined with peramivir at 0.25 and 0.5 mg/kg/d. These data demonstrate that combinations of favipiravir and peramivir perform better than suboptimal doses of each compound alone for the treatment of influenza virus infections in mice.     

The effect of disodium 4-chloro-2-iminodibenzoate (CCA) on IgE levels and anaphylactic shock

The effect of disodium 4-chloro-2,2-iminodibenzoate (CCA) on IgE antibody response was examined in C3H/A and (BALB/c x C57BL/6J) F1 hybrid mice immunized with low doses of ovalbumin (OA) adsorbed on aluminium hydroxide gel. CCA administered orally at the doses of 5 and 50 mg/kg/day reduced IgE antibody production in these mice as determined by PCA test. High doses of CCA (100 mg/kg/day) given from day 7 before immunization of C57BL mice and during 1 week after immunization of mice with OA and Bordetella Pertussis Vaccine reduced the mortality of these mice subjected to anaphylactic shock on day 7 of immunization. CCA treatment was ineffective in anaphylactic shock of C57BL mice immunized with very high dose of OA, known to elicit little or no IgE antibody production but high IgG antibody response. The treatment of OA-immunized Guinea pigs with one oral dose of CCA (100 mg/kg) did not reduce mortality in protracted anaphylactic shock. Our results demonstrate that CCA inhibits IgE production as well as IgE mediated hypersensitivity reactions in mice.     

Effects of acute administration of O,S,S-trimethyl phosphorodithioate on the generation of cellular and humoral immune responses following in vitro stimulation

The time course of immune modulation induced by acute treatment with O,S,S-trimethyl phosphorodithioate (OSS-TMP), an impurity in technical formulations of malathion, was examined in female C57BL/6 mice. The immune parameters studied included the generation of cytotoxic T lymphocytes (CTL) to alloantigen (H-2 incompatible) and antibody secreting cells to sheep red blood cells, proliferative response to the mitogens, and interleukin-2 (IL-2) production. Acute administration of the non-toxic doses of OSS-TMP, i.e. 20 or 40 mg/kg, led to an elevation in the generation of a CTL response on day 1 or 7, respectively. At 20 mg/kg OSS-TMP, the antibody response was elevated at day 3. However, at a dose of 40 mg/kg OSS-TMP, the antibody response was suppressed at day 1 following treatment. Following acute administration of 60 or 80 mg/kg OSS-TMP, the generation of an antibody and CTL responses was suppressed at all time points tested with 1 exception. One day following treatment at a dose of 60 mg/kg OSS-TMP, there was no change in the CTL response. At day 7 following treatment, the mitogenic responses to lipopolysaccharide and phytohemagglutinin were elevated at all doses of OSS-TMP administered. At this time point, however, the proliferative response to Concanavalin A was elevated in a dose dependent manner. IL-2 production was suppressed following acute administration of 60 or 80 mg/kg OSS-TMP at all time points tested and at all doses tested on day 5 following treatment. These data indicate that OSS-TMP, unlike its congener, O,O,S-trimethyl phosphorothioate, enhances the generation of humoral and cell mediated immune responses of C57BL/6 mice following administration of non-toxic doses.     

Manipulation of mouse organ glutathione contents. II: Time and dose-dependent induction of the glutathione conjugation system by phenolic antioxidants

After 14 days of oral butylated hydroxyanisole (BHA) administration (1000 mg/kg/day) the tissue glutathione levels of male NMRI mice were increased by 74-141% in liver, lung, duodenum and intestine and after similar butylated hydroxytoluene (BHT) treatment by 18-85% in the liver, lung, spleen and the gastrointestinal tract. Doses of 100 mg/kg/day significantly elevated the glutathione content in the lung (BHA, BHT), duodenum (BHA) and intestine (BHA), while 10 mg/kg/day affected only lung glutathione content (BHA). BHA treatment (1000 mg/kg/day) induced GST activities significantly (138-1335%) in all organs investigated except the spleen, i.e. liver, lung, kidney and the entire gastrointestinal tract, while a similar dose of BHT increased GST activities in the liver, duodenum, intestine and colon by 26-339%. Daily doses of 100 mg/kg/day significantly induced GST activities only in the liver (BHA, BHT), lung (BHA) and kidney (BHA). Lower doses of BHA or BHT did not significantly affect GST activities in the organs investigated (except 10 mg BHA/kg/day in the lung). Comparison of the time course of induction of the glutathione conjugation system in various organs after different doses of antioxidants indicated no change between 5 and 14 days of treatment with all doses used (1-1000 mg/kg). Only the lung glutathione level showed a tendency to increase with low dose BHA by extending the time of treatment. The time course of the liver glutathione content between single doses of 100 mg/kg BHA or BHT revealed an initial decline followed by an increase above control values 2 days (BHA) or 5 days (BHT) after the first application. The glutathione levels of the lung and the duodenum increased without a preceding decline. Only the second dose of BHT caused a temporary decrease to control values of the elevated glutathione level in the duodenum. All animals (at any dose of BHA or BHT) showed control values of serum transaminase activities. These results suggest: The induction threshold of the glutathione conjugation system in various mouse organs is greater than or equal to 100 mg/kg for BHA and BHT. Chronic administration of these compounds did not change these results (except the lung glutathione level after low dose BHA). Elevated hepatic glutathione levels might be the result of an activated synthesis caused by a preceding loss of glutathione. Chronic BHA or BHT treatment did not cause hepatotoxic effects, as evaluated by serum transaminases, in male mice.     

Oral administration of exopolysaccharide from Aphanothece halophytica (Chroococcales) significantly inhibits influenza virus (H1N1)-induced pneumonia in mice

The halophilous cyanobacterium Aphanothece halophytica releases large sums of single type sulfated exopolysaccharide in late logarithmic growth phase in culture. This polysaccharide contained sulfate up to 34.46% of the total moieties in the molecular. As a sulfated polysaccharide that can be biosynthesized in large quantities, however, its antiviral activity has not yet been reported. In this study, we examined effects of exopolysaccharide from A. halophytica Fremy (EPAH) on influenza virus A FM (H1N1) (FM1)-induced pneumonia and reduction in immunocompetence in mice. Previous and simultaneous treatment of EPAH at a dose of 60 mg/kg significantly inhibited pneumonia in FM1-infected mice by 30.4% and 26.7%, respectively. In post-treatment, EPAH displayed its most effective inhibition at a dose of 80 mg/kg with the inhibition rate at 18.69%. Simultaneous treatment of FM1-infected mice with EPAH showed effective improvement on reduction of lymphocyte number with its most effective dose at 60 mg/kg. FM1-infected mice simultaneously received EPAH at a dose of 40 mg/kg also acquired obvious enhancement on release of IL-2 on day 15, and those received EPAH at a dose of 60 mg/kg showed similar enhancement on day 10. Simultaneous treatment with EPAH indicated remarkable recovery or improvement of FM1-induced reduction of IL-1beta level and phagocytic capacity of RES. Simultaneous treatment with EPAH significantly resumed the cytolytic activity of natural killer cells in FM1-infected or CP treated mice at doses of 40 and 60 mg/kg. These results suggested that EPAH is an effective agent against FM1. The mechanisms of its action might be mediated, at least in part, by modulating the host immune system and the interaction positive charges in EPAH and negative charges FM1.     

Dose-Dependent Pharmacokinetics of the Aldose Reductase Inhibitor Imirestat in Man

The pharmacokinetics of imirestat were studied in healthy volunteers following single and multiple oral doses. After single doses of 20 to 50 mg, imirestat plasma concentrations declined with an apparent elimination half-life of 50 to 70 hr over the 168 hr in which levels were measured. However, with lower doses (2 to 10 mg), an initial rapid decline in drug concentration was followed by a very slow terminal elimination phase with plasma concentrations decreasing little over the 1 week of sampling. This resulted in a decrease in apparent t 1/2 with increasing dose, from 272 +/- 138 hr at 2 mg to 66 +/- 30 hr at 50 mg. During once-daily dosing of 2 to 20 mg/day for 4 weeks, mean steady-state imirestat concentration appeared to be dose proportional, although the time required to achieve steady state decreased with increasing dose. The mean effective half-life for accumulation ranged from 54 to 98 hr, suggesting that the very slow elimination of drug at low concentrations did not produce disproportionate accumulation of drug at these doses. Mean oral clearance was independent of dose, ranging from 30 to 45 ml/min. At the 2-, 5-, and 20-mg doses, one subject in each group had steady-state concentrations two- to fourfold greater than any of the other five subjects at the same dose, although the reason for this was not apparent from these data. The overall kinetic profile of these data was suggestive of dose-dependent pharmacokinetics resulting from nonlinear tissue binding of imirestat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute Hepatotoxic and Nephrotoxic Effects of Chloroform in Male F-344 Rats and Female B6C3F1 Mice

Previous studies demonstrated that chloroform given by oralgavage in corn oil caused an increased incidence of liver tumorsin male and female mice and kidney tumors in male rats, whileadministration in drinking water resulted in an increased tumorincidence only in the kidneys of the male rats. The tumorigenicityof this nongenotoxic agent has been postulated to be linkedwith cytolethality and cell proliferation. This study examinedthe organ-specific toxicity of acute doses of chloroform. MaleF-344 rats were given chloroform by gavage in corn oil at thebioassay doses of chloroform of 0 and 180 mg/kg body wt as wellas 34 and 477 mg/kg and necropsied 24 hr later. Additional ratswere given a single dose of 180 mg chloroform/kg and administeredbromodeoxyuridine (BRDU) 2 hr prior to necropsy at 0.5, 1, 2,4, and 8 days after chloroform treatment. Female B6C3F1 micewere given chloroform by gavage at the bioassay doses of 0,238, and 477 mg/kg as well as 34 mg/kg and necropsied at 24hr after treatment. Additional mice were given a single doseof 350 mg chloroform/kg, labeled with BRDU, and necropsied at0.5, 1, 2, 4, and 8 days after treatment. The kidneys of malerats administered 34, 180, and 477 mg chloroform/kg exhibitedmild to severe proximal tubular necrosis in a dose-dependentmanner. A 20-fold increase in the labeling index (LI, the percentageof nuclei in S-phase) in the proximal tubule cells was observed2 days after treatment with the bioassay dose of 180 mg/kg.The livers of male rats exhibited only slight to moderate multifocalcentrilobular necrosis at 180 and 477 mg/kg. A 10-fold increasein the LI was observed in the liver of male rats given 477 mg/kg,but no increase was observed at the bioassay dose of 180 mg/kg.In contrast to male rats, female mice developed a dose-dependentcentrilobular hepatic necrosis at 238 and 477 mg/kg. No renallesions were observed in female mice at any dose. A peak increasein LI of 38-fold was observed in hepa-tocytes in the liversof female mice 2 days after treatment with 350 mg chloroform/kg,with only a 2-fold increase in LI observed in the kidneys. Thesedata indicate that acute chloroform-induced cytolethality leadsto increased cell proliferation and that the organ-specificpattern of toxicity is the same as the organ-specific patternof tumor formation. These observations are suggestive of a rolefor induced cell proliferation in tumor formation and indicatethat more extensive cell proliferation Studies are Warranted.     

Toxicological Evaluation of the Cardiotonic Isomazole in the Dog

Acute, subchronic, and chronic toxicity studies were conductedin dogs with the new vasodilator/cardiotonic drug isomazole(IMZ) to support, in part, clinical investigations of this agentin humans. Single oral doses of IMZ of 25, 50, or 100 mg/kggiven to English pointer dogs (2/dose) caused a marked dropin systemic blood pressure and reflex-induced increases in heartrate to values well over 200 beats per minute. These responseswere maintained for 12 to 22 hr depending on the dose given.One of the dogs receiving 100 mg/kg died at 4.5 hr postdose.Results of subchronic (3 months) and chronic (1 year) studiesin beagle dogs (4/sex/dose group), in which measurable plasmalevels of the drug and its metabolites were found, indicatedthat IMZ did not produce any discernible adverse findings whengiven in doses up to 16 mg/kg, other than expected cardiotoxiceffects. The plasma t_1/2 of IMZ at 16 mg/kg increased to between4 and 8 hr from 2 hr noted at lower doses. In the 1-year study,at all doses and in both sexes, plasma levels of IMZ declinedover the first month, stabilizing (at the 2 and 6 mg/kg doses)thereafter for the duration of the study. At the high dose of16 mg/kg, after 1 year plasma levels of IMZ exceeded (females)or equaled (males) the 1-month values. At peak plasma levelsof IMZ (2 hr postdose), plasma levels of parent drug increasedlinearly with the dose. The cardiotoxic effects consisted ofsubstantial postdose increases in heart rate throughout thecourse of treatment (5 mg/kg and above), significant increasesin heart weight (6 mg/kg and above), and multifocal myocardialfibrosis (6 mg/kg and above). There was a decline in basal heartrate at doses of 12.5 mg/kg and higher. The results of thesestudies demonstrated that repeated IMZ administration, as expected,was cardiotoxic to the dog, a species relatively sensitive tothe pharmacological activity and hemodynamic changes inducedby vasodilator/cardiotonic drugs. The no-effect dose level forcardiotoxicity in the repeated dose studies was considered tobe 2 mg/kg, the lowest dose tested.     

The discriminative stimulus properties of methylphenidate in C57BL/6J mice

Methylphenidate (MPH) therapy for attention-deficit/hyperactivity disorder is common in children and adults. Concerns regarding abuse of MPH prompted studies to better understand its pharmacology. We used an established drug discrimination task to determine whether MPH could be discriminated by C57BL/6J (B6) mice. B6 mice learned to discriminate cues produced by racemic MPH (dl-MPH 5.0 mg/kg) or half the dose of pure d-isomer (2.5 mg/kg), and dose-response tests established appropriate reductions in discrimination with declining dose. Importantly, the two drug forms generalized to each other completely in substitution tests; consistent with reports that the l-isomer is pharmacodynamically inactive. An additional experiment indicated that lower doses (1 and 2 mg/kg) of dl-MPH did not support acquisition of MPH discrimination despite extensive training. Mice acquired discrimination of dl-MPH only when the dose was increased to 4 mg/kg. Thus, although these lower doses increased drug lever responding in mice trained on the higher dose, their stimuli were not sufficient to support acquisition of the discrimination task. These findings correspond to earlier studies conducted in our laboratory on threshold doses needed to produce stimulatory effects of motor activity in B6 mice. These preclinical findings provide insight into the relative potency, and by extension, efficacy of dl-MPH versus d-MPH doses.