Editorial comment

A single injection of Cyclophosphamide (CY) in a dose of 300 mg of CY/kg of mice resulted in enhanced delayed hypersensitivity (DH) when administered between at least 7 days prior to and 15 days after intracutaneous (ic) immunization of sheep red blood cells in Freund's complete adjuvant. The maximal enhancement occurred when CY was applied 8 hr before the antigen. Using the latter interval, the effect of varying the dose of CY before ic or intraperitoneal (ip) injections of antigen was studied. Combined with ic immunization, increasing doses of CY resulted in increasing DH. The ip route of immunization needed CY in amounts of at least 100 mg/kg to augment DH to a detectable level. The enhancing effect of lower doses of CY was more pronounced when the interval between immunizing and eliciting injections was reduced. Administration of 300 or 200 mg of CY/kg before ip immunization suppressed the antibody formation, when measured S and 7 days later. A dose of 100 mg of CY/kg caused a suppression of antibody formation on Day 5, but an enhancement on Day 7. With this dose, a maximal enhancement of DH was found on both days. The results suggest that CY interferes with more than one regulatory mechanism of the immune response.     

The effects of cyclophosphamide on the toxicity and immunogenicity of ricin A chain immunotoxin in rats

We conducted a study to determine if treatment with cyclophosphamide (CY) could suppress the formation of anti-murine and anti-ricin A chain antibodies in rats treated with a murine monoclonal antibody-ricin A chain immunotoxin (IT). Female Sprague-Dawley rats received intravenous doses of IT at a dose of 5 mg/kg body weight alone or in combination with CY at a dose level of either 10 or 20 mg/kg body weight. The IT was given as one or two courses consisting of five consecutive daily intravenous injections (days 0 to 4, or days 0 to 4 and days 21 to 25 of the study). Cyclophosphamide was given on days 2, 4, 6, 13, and 17 of the study to the group receiving a single course of IT; additional doses of CY were administered on days 23, 25, and 27 to the group receiving two courses of IT. On days 4, 14, 21, 28, and 35, animals from each group were evaluated for antibodies to murine IgG and ricin A chain, and for clinical laboratory parameters and histopathology. Animals receiving IT alone developed significant titers of both anti-murine and anti-ricin A chain antibodies. Compared with the response in the animals receiving single-course IT, the response to both of the components of the IT was significantly increased on days 28 and 35 in the animals receiving a second course of IT. The groups receiving a combination of either one or two courses of CY and IT demonstrated a significantly decreased antibody response to both the murine IgG and the ricin A chain compared with the group receiving IT alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary selenium on the response of stressed and unstressed chickens toEscherichia coli challenge and antigen

Selenium was added to the feed of White Leghorn type chickens 1 day prior to challenge with eitherEscherichia coli or sheep erythrocyte antigen. the incidence of death or lesions was reduced from 86% to 21% at the optimal dose of selenium (0.4 mg/kg resulting in feed concentration of 0.45 mg/kg). After the chickens were stressed by chilling, selenium was ineffective againstE. coli. Dietary additions of selenium between 0.1 and 0.8 mg/kg resulted in an antibody titer increase from 2.2 to 3.9 to the log₂ against sheep erythrocytes (SRBC). Followng chilling, antibody titer response was reduced from 4.9 to 2.4 to the log₂. This titer reduction could be prevented with dietary additions of selenium between 0.1 and 1.2 mg/kg. The effects of a nitrofuran and selenium were additive againstE. coli challenge infection.     

Foreign serum-induced bile duct lesion (BDL) in athymic BALB/c nude mice

To investigate a role of cellular immunity in foreign serum-induced bile duct lesion (BDL) in mice, athymic BALB/c nude (nu/nu) mice were intraperitoneally injected with swine serum (SS) twice a week up to 8 weeks and were compared with euthymic BALB/c heterozygote (nu/+) and wild-type (+/+) mice treated with SS in the same way for 4 weeks. All immunized nu/+ and +/+ mice developed marked BDL, and their sera showed high anti-SS IgE and IgG1 antibody titers, whereas no immunized nu/nu mice developed lesions, and their sera showed no elevation of antibody titers. Next, nu/nu mice were reconstituted with splenocytes derived from nu/+ mice, and then were intraperitoneally injected with SS twice a week for 3 weeks. Most of the reconstituted nu/nu mice developed BDL, and their sera showed the elevation of anti-SS IgE and IgG antibody titers. These results suggest that cellular immunity may play a pivotal role in the pathogenesis of swine serum-induced BDL.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Interference of anti-tumor and immunosuppressive effects of cyclophosphamide in tumor-bearing rats

Summary Adult rats were given 10⁵ or 10⁶ Yoshida ascites sarcoma (YAS) cells IP and were treated with cyclophosphamide (CY) given IP in single doses of 20 mg/kg or 100 mg/kg, 2 or 5 days after YAS inoculation. Both the curative effect of CY and subsequent resistance to tumor challenge in rats that survived depended on the dose of injected tumor cells and on the dose and time of administration of CY. These three factors determined whether the host's immune response to tumor antigens would develop and contribute to the overall anti-tumor effects of the chemotherapy. The curative effects of CY were significantly less pronounced in T-cell-deficient than in normal rats. Anti-tumor and immunosuppressive activities of CY exerted opposite influences on the ultimate result of the chemotherapy. Adverse immunosuppressive effects prevailed when the drug was administered early (2 days) after YAS inoculation. In this case the chemotherapy was less efficient and the surviving rats were susceptible to a subsequent tumor challenge. Further analysis showed that the injection of CY 2 days after inoculation of YAS antigens induced strong and specific immunologic tolerance to the tumor. In contrast, when a sufficient amount of tumor antigens (higher dose of tumor cells injected and CY injection delayed) elicited an anti-YAS immune response that was not suppressed by early injection of CY (CY administered 5 days after the tumor) effective eradication of tumor cells and anti-YAS resistance in cured animals were observed.Abbreviations YAS Yoshida ascites sarcoma - CY cyclophosphamide - MRBC mouse red blood cells     

Morphological changes in the endocrine pancreas of mice after treatment with streptozotocin combined with cyclophosphamide and neurotropin.

The effect of neurotropin (NSP) in combination with streptozotocin (STZ) and cyclophosphamide (CY) on blood glucose and pancreatic histopathology on day 7 and day 14 after the initiation of the treatment was studied in C57Bl/6 male mice. STZ (40 mg/kg) and NSP (1 mg/kg) were applied intraperitoneally on five consecutive days and CY (150 mg/kg)--twice on day 1 and day 3. In single B cells dilatation of the endoplasmic reticulum was found. On day 7 in proximity to some endocrine cells in the mice treated with STZ, STZ + CY + NSP and STZ + CY macrophages were observed. On day 14 lymphocytic infiltration of the islets was demonstrated only in the groups of mice injected with STZ, STZ + CY while in the group treated with the combination STZ + CY + NSP no infiltration was seen. All experimental groups showed no biochemical evidence for hyperglycemia probably due to the mild destruction of a small number of B cells. The results indicate that NSP might possess a restorative action on insulitis induced by multiple low dose streptozotocin administration in mice.     

姜黄素对小鼠胆汁淤积性肝纤维化的改善作用及其机制研究*

目的:探讨姜黄素对小鼠胆管结扎所致的胆汁淤积性肝纤维化的保护作用,为肝纤维化治疗提供新的治疗方法。方法:42只健康成年雄性BALB/c小鼠随机分为假手术(n=6)处理组、假手术+姜黄素(n=6)处理组、胆管结扎(BDL)处理组(n=10)、BDL+姜黄素处理组(n=10),BDL+姜黄素+锌原卟啉(ZnPP)处理组(n=10)。BDL手术7 d后,假手术+姜黄素组、BDL+姜黄素组每日给予姜黄素(30 mg / kg)腹腔注射;BDL+姜黄素+ZnPP组每日给予姜黄素(30 mg / kg)以及nPP(50 μmol/ kg)腹腔注射;对于假手术组和BDL组,小鼠每天一次腹膜内注射等体积的盐水。整个给药过程持续7 d。小鼠BDL14 d后,取血和肝脏组织,检测谷草转氨酶(AST)、谷丙转氨酶(ALT)水平,观察肝组织病理形态变化、肝纤维化情况、检测肝组织中血红素加氧酶-1(HO-1)的蛋白表达。结果:与假手术组相比,BDL组小鼠肝脏胆囊肿大,血清谷草转氨酶(ALT)、谷丙转氨酶(AST)水平显著升高 (P＜0.05),同时,天狼星红染色及促纤维化相关基因的qRT-PCR结果显示肝脏出现胶原蛋白沉积,巨噬细胞及中性粒细胞免疫组化结果显示肝脏出现炎性细胞浸润;与BDL组相比,姜黄素治疗组血清ALT、AST水平明显降低(P＜0.05),胶原蛋白沉积及炎性细胞浸润情况有所改善,同时,补充姜黄素后HO-1表达升高(P＜0.05);对姜黄素治疗组给予HO-1活性抑制剂ZnPP发现,姜黄素对肝损伤的保护作用被逆转。结论:姜黄素可以改善BDL所致的肝脏炎症及肝纤维化,这种保护作用可能与姜黄素调节HO-1活性有关。     

注射Bestatin对幼年大鼠分辨学习的影响

以Ｓｐｒａｑｕｅ－Ｄａｗｌｅｙ大鼠为实验动物,从出生后第１天起,每日皮下分别注射氨基肽酶的抑制剂Ｂｅｓｔａｔｉｎ（１ｍｇ／ｋｇ（ｂ．ｗ））以及钠络酮（２ｍｇ／ｋｇ（ｂ．ｗ）＋Ｂｅｓｔａｉｎｇ（１ｍｇ／ｋｇ（ｂ．ｗ））,对照组注射等量的生理盐水,连续注射１４天,观察：１６日龄幼鼠的吸乳迷津分辨学习（ＡＤＬ）,３０日龄Ｙ迷津明暗分辨学习（ＡＤＬ）,３０日龄Ｙ迷津明暗分辨学习（ＢＤＬ）行为,４５日龄幼鼠     

Effects of the antimicrobial peptide BMAP-27 in a mouse model of obstructive jaundice stimulated by lipopolysaccharide

An experimental study was designed to investigate the efficacy of BMAP-27, a compound of the cathelicidin family, in neutralizing Escherichia coli 0111:B4 lipopolysaccharide (LPS) in bile duct-ligated mice. Main outcome measures were: endotoxin and TNF-alpha concentrations in plasma, evidence of bacterial translocation in blood and peritoneum, and lethality. Adult male BALB/c mice were injected intraperitoneally with 2 mg/kg E. coli 0111:B4 LPS 1 week after sham operation or bile duct ligation (BDL). Six groups were studied: sham with placebo, sham with 120 mg/kg tazobactam-piperacillin (TZP), sham with 1 mg/kg BMAP-27, BDL with placebo, BDL with 120 mg/kg TZP, and BDL with 1mg/kg BMAP-27. After LPS, TNF-alpha plasma levels were significantly higher in BDL mice compared to sham-operated animals. BMAP-27 achieved a significant reduction of plasma endotoxin and TNF-alpha concentration when compared with placebo- and TZP-treated groups. On the other hand, both TZP and BMAP-27 significantly reduced the bacterial growth compared with saline treatment. Finally, LPS induced 60% and 55% lethality in BDL placebo- and TZP-treated treated mice and no lethality in sham-operated mice, while only BMAP-27 significantly reduced the lethality to 10%. In light of its dual antimicrobial and anti-endotoxin properties, BMAP-27 could be an interesting compound to inhibit bacterial translocation and endotoxin release in obstructive jaundice.     

Contrary action of khingamin on the rejection of a skin allograft and on the level of antibody-producing cells in mice

The effect of antimalarial drug khingamin preinjection on skin allograft rejection and the level of antibody forming cells (AFC) in Jerne reaction has been investigated. The drug at the doses of 30, 40, 50 mg/kg inhibited skin allograft rejection. The dose of 20 mg/kg proved ineffective. A single preinjection at a dose of 20 mg/kg had a stimulating effect on antibody formation, whereas the dose of 40 mg/kg induced no effect. When the latter dose was injected simultaneously with sheep red blood cells, it caused a significant inhibition of AFC levels. Fourfold preliminary injection of khingamin at doses 40 and 20 mg/kg had a stimulating effect. The reverse effect of khingamin may be accounted for by the diverse action of the drug itself and its metabolites.     

Relationship between experimental results in mammals and man. I. Cytogenetic analysis of bone marrow injury induced by a single dose of cyclophosphamide.

The extrapolation of experimental results to man was studied by cytogenetic bone marrow analysis and micronucleus test in mice, rats and Chinese hamsters. Furthermore, the frequency of chromosomal aberrations was compared with the frequencies of polychromatic erythrocytes containing micronuclei. Cyclophosphamide (CY) was given intraperitoneally at the doses of 5, 10, 20, 40 and 80 mg/kg b.w. to ICR mice and Wistar rats and at the doses of 10, 20, 40, 80, 120 and 160 mg/kg b.w. to Chinese hamsters. Five patients with various types of malignancies until then medically untreated, were i.v. administered 40 mg CY/kg b.w. Bone marrow cells were examined 24 h after the administration. CY induced in all rodents a clear-cut dose-effect relationship in the frequency of breaks, abnormal metaphases as well as in the frequency of micronuclei in polychromatic erythrocytes. When comparing the results in rodents and man at the dose of 40 mg CY/kg b.w., the sensitivity pattern of species was mice greater than rats greater than Chinese hamsters greater than man. From this aspect the possible differences in the metabolism of CY in analysed species are discussed. The presented results tend to a conclusion that micronucleus testing may be a very suitable method used for screening purpose, however, the method of classical cytogenetic analysis, especially the evaluation of breaks, still remains the most exact and reliable technique.     

Effects of Dietary Form of Selenium on Its Distribution in Eggs

The objective of this experiment was to investigate the selenium distribution in eggs from hens fed diets supplemented with Se from sodium selenite (SS) or selenium-enriched yeast (SY). One-day-old female chickens of Hy-Line Brown breed were randomly divided into four groups according to dietary treatments and, for the subsequent 9?months, were fed diets which differed only in the form or amount of Se supplemented. During the whole experiment, group 1 (control) was fed basal diet (BD) with only background Se level of 0.13?mg/kg dry matter (DM). Diets for groups 2 and 3 consisted of BD supplemented with an Se dose of 0.4?mg/kg DM either in the form of SS or SY, respectively. Group 4 was fed BD supplemented with 0.9?mg Se/kg DM from SY. After 9?months of dietary treatments, the Se levels in egg yolk and albumen from hens fed unsupplemented diet were almost identical whereas eggs from hens given diet supplemented with SS showed significantly higher Se deposition in yolk than in albumen (P?相似文献   

Single-dose murine monoclonal antibody ricin A chain immunotoxin in the treatment of metastatic melanoma: a phase I trial.

To determine the maximally tolerated dose of a ricin A chain-conjugated antimelanoma antibody (XomaZyme-Mel), 20 patients with metastatic melanoma were treated with escalating doses of the murine immunotoxin given as single intravenous infusion over 30 minutes. The starting dose was 0.6 mg/kg and was escalated in five groups to a maximum of 1.6 mg/kg. The maximally tolerated dose was 1.25 mg/kg as three of six patients treated at 1.6 mg/kg developed unacceptable toxicity. The dose-limiting toxicity consisted of profound fatigue, myalgias, and arthralgias. These occurred within 4 days and resolved in 7 to 10 days. Other non-dose-limiting toxicities encountered consisted of hypoalbuminemia, weight gain, peripheral edema, mild hypotension, and flu-like syndrome; the severity of these was also dose related. In addition, two allergic reactions occurred, one severe. There was one durable complete response of 12+ months' duration and one brief mixed response lasting 3 months. We conclude that the maximum tolerated single dose of XomaZyme-Mel is 1.25 mg/kg. Phase I studies evaluating 1.25 mg/kg given in multiple doses at 2- to 4-week intervals and phase II studies to determine the response rate of a single 1.25 mg/kg dose are warranted.     

A correlation between conditioning and engraftment in recipients of MHC-mismatched T cell-depleted murine bone marrow transplants

We studied engraftment in a murine model of allogeneic bone marrow (BM) transplantation. Recipient C57BL/6 (H-2b) mice were conditioned with single-dose (9 or 7.5 Gy) total body irradiation (TBI), fractionated (4 X 3.3 Gy) TBI, hyperfractionated (8 X 1.65 Gy) TBI, 2 X 120 mg/kg cyclophosphamide (CY) followed by 7.5 Gy TBI, or 300 mg/kg CY followed by 9 Gy total lymphoid irradiation (TLI). Conditioned mice were transplanted with BALB/c (H-2d) BM supplemented with splenocytes (BMS) to facilitate graft-vs-host disease (GVHD). Ex vivo T cell depletion of the BMS with anti-Thy-1.2 antibody and complement protected recipients from lethal GVHD. Engraftment was measured in transplanted animals by serotyping peripheral blood mononuclear cells with anti-H-2-specific antibodies and complement. Mice that were given a T cell-depleted BMS transplant after conditioning with 9 Gy TBI, fractionated TBI, or CY plus TBI showed a 99 to 100% incidence of engraftment. However, if the T cell-depleted graft was given to mice conditioned with hyperfractionated TBI, 7.5 Gy TBI, or CY plus TLI, only 3 to 32% of the animals engrafted. BM which was not T cell-depleted engrafted in 63 to 100% of the mice regardless of the conditioning used. Nonengrafted mice tested with anti-host type antibody demonstrated autologous recovery. We conclude that engraftment or failure/rejection of BM in transplanted mice is determined in part by a dynamic equilibrium between T cells present in the donor graft and the surviving hemopoietic cells in the conditioned recipient. More intensive conditioning of the recipient allows engraftment of T cell-depleted, mismatched BMS. Such conditioning is not limited to a single modality, but can be achieved with single-dose TBI, fractionated TBI, or with TBI combined with CY. These findings have timely and important implications for the current understanding of engraftment in human allogeneic BM transplantation following T cell depletion.     

An Enhanced Pre‐ and Postnatal Development Study in Cynomolgus Monkeys with Tabalumab: A Human IgG4 Monoclonal Antibody

Tabalumab, a human IgG4 monoclonal antibody (mAb) with neutralizing activity against both soluble and membrane B‐cell activating factor (BAFF), has been under development for the treatment of autoimmune diseases. The purpose of this study was to determine the potential adverse effects of maternal tabalumab exposure on pregnancy, parturition, and lactation of the mothers and on the growth, viability, and development of the offspring through postnatal day (PND) 204. Tabalumab was administered by subcutaneous injection to presumed pregnant cynomolgus monkeys (16–19 per group) every 2 weeks from gestation day (GD) 20 to 22 until parturition at doses of 0, 0.3, or 30 mg/kg. Evaluations in mothers and infants included clinical signs, body weight, toxicokinetics, blood lymphocyte phenotyping, T‐cell‐dependent antibody response (infants only), antitherapeutic antibody (ATA), organ weights (infants only), and gross and microscopic histopathology. Infants were also examined for external and visceral morphologic and neurobehavioral development. There were no adverse tabalumab‐related effects on maternal or infant endpoints. An expected pharmacological decrease in peripheral blood B‐lymphocytes occurred in adults and infants; however, B‐cell recovery was evident by PND154 in adults and infants at 0.3 mg/kg and by PND204 in infants at 30 mg/kg. At 30 mg/kg, a reduced IgM antibody response to T‐cell‐dependent antigen keyhole limpet hemocyanin (KLH) was observed following primary immunization. Following secondary KLH immunization, all infants in both dose groups mounted anti‐KLH IgM and IgG antibody responses similar to control. Placental and mammary transfer of tabalumab was demonstrated. In conclusion, the no‐observed‐adverse‐effect level for maternal and developmental toxicity was 30 mg/kg, the highest dose tested. Exposures at 30 mg/kg provide a margin of safety of 16× the anticipated clinical exposure.     

TGF‐β1 monoclonal antibody: Assessment of embryo‐fetal toxicity in rats and rabbits

A humanized monoclonal antibody targeting transforming growth factor β1 (TGF‐β1 mab) has been used in development for the treatment of chronic kidney disease. Embryo‐fetal development studies were conducted in rats and rabbits using 30 and 25 animals per group, respectively. The TGF‐β1 mab was administered subcutaneously to rats at 0, 2, or 50 mg/kg/dose on gestation days (GDs) 6, 10, and 14 and intravenously to rabbits at 0 or 3 mg/kg/dose on GDs 7, 12 to 19, and at 30 mg/kg/dose on GDs 7, 12, 14, 16, and 18. Maternal reproductive endpoints and fetal viability, weight, and morphology were evaluated. There was no indication of maternal or embryo‐fetal toxicity in the rat. Effects in the rabbit were limited to the fetus where the 30 mg/kg TGF‐β1 mab dose produced a slight decrease in fetal weight and an increase in the incidence of retrocaval ureter and an absent and/or malpositioned kidney/ureter in two fetuses. In conclusion, TGF‐β1 mab produced no adverse maternal or embryo‐fetal findings in rats when administered ≤50 mg/kg on GDs 6, 10, and 14. TGF‐β1 mab did not demonstrate maternal toxicity or embryo‐fetal lethality at doses as high as 30 mg/kg when administered on GDs 7, 12, 14, 16, and 18 in rabbits. Fetal growth and morphology were affected only at 30 mg/kg; thus, the no observed adverse effect level was 3 mg/kg in rabbits. The margin of safety for both rats and rabbits was ≥37‐fold the clinical exposure level.     

Preliminary immunomodulatory activities of methanol extracts of Eclipta alba and Centella asiatica.

An attempt has been made to assess the immunomodulatory activity of methanol extracts of whole plant of E. alba (1.6% wedelolactone) and C. asiatica (0.18% of asiaticoside) at five dose levels (dose-response relationship) ranging from 100 to 500 mg/kg body wt. using carbon clearance, antibody titer and cyclophosphamide immunosuppression parameters. In the case of E. alba, the phagocytic index and antibody titer increased significantly and the F ratios of the phagocytic index and WBC count were also significant. Regression analysis showed linearity in patterns of the dose-response relationship, greatest in the case of the phagocytic index, moderate in the WBC count and lowest in the antibody titer. For C. asiatica, significant increases in the phagocytic index and total WBC count were observed and the F ratio of the phagocytic index was also significant. Regressed values revealed maximum linearity in the case of the phagocytic index, moderate linearity in the total WBC count and lowest linearity in the antibody response.     

Ameltolide. I: Developmental toxicology studies of a novel anticonvulsant

Ameltolide, a novel anticonvulsant agent, has been shown in animal models to be effective in controlling seizures. The developmental toxicity of ameltolide was evaluated in two species. Naturally mated rats and rabbits were dosed once daily by gavage on gestation days (GD) 6-17 and 6-18, respectively. Rats were given doses of 0, 10, 25, or 50 mg/kg; rabbits were given 0, 25, 50, or 100 mg/kg. Laparotomy was performed on rats on GD 20 and on rabbits on GD 28. In rats, maternal toxicity was indicated at the 25- and 50-mg/kg dose levels by depressed body weight gain. Fetal body weight was depressed at the 50-mg/kg dose level. Fetal viability and morphology were not affected. The no-observed effect levels (NOEL) for adult and developmental toxicity in the rat were 10 and 25 mg/kg, respectively. In rabbits, maternal toxicity was indicated by a net loss in body weight at the 50- and 100-mg/kg dose levels. Fetal viability and body weight were depressed at the 100 mg/kg dose level. Shortened digits occurred on the right forepaw of one fetus at the 50-mg/kg dose level (in conjunction with severe maternal toxicity) and on the hindpaws of two fetuses from separate litters at the 100-mg/kg dose level. Incomplete ossification of the phalanges occurred on the forepaws of nine fetuses from four litters at the 100-mg/kg dose level. Ameltolide was weakly teratogenic in the rabbit. The NOEL for adult and developmental toxicity in the rabbit was 25 mg/kg.     

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of rats exposed to 2-acetylaminofluorene (2AAF)

Groups of male Alderley Park rats were dosed concomitantly with 2-acetylaminofluorene (2AAF) by gavage at doses between 0.01 mg/kg and 40 mg/kg, and livers sampled 2-72 h later. The liver of one group of animals was perfused to yield hepatocytes which were assayed in vitro for unscheduled DNA synthesis (UDS) via incorporation of tritiated thymidine and autoradiography. DNA was extracted from the livers of the other group and DNA adduct levels determined using the 32P-postlabelling technique. The major C-8 2-aminofluorene/guanosine adduct and 3 minor adducts were quantitated, enabling the relative sensitivity of the 2 techniques to be compared. A dose- and time-related UDS response was observed, which, at the most sensitive time-point (12 h) enabled DNA repair to be discerned at a dose level of 0.1-1 mg/kg of 2AAF, a response classified as formally positive at 5 mg/kg 2AAF. Only the C-8 adduct, as determined by 32P-postlabelling, was discernible at 0.01 mg/kg of 2AAF, although other adducts were visible on autoradiograms at higher dose levels. It is concluded that as part of a well-defined dose response, UDS can be discerned with confidence for doses of 2AAF between approximately 0.1 and 5 mg/kg, and DNA adducts for doses of 2AAF between approximately 0.01 and 1 mg/kg. Discernible UDS for 2AAF in the rat liver is apparent at approximately 13 DNA (total) adducts/10(8) nucleotides, or approximately 8 DNA (C-8) adducts/10(8) nucleotides. The presumed C-8 2-acetylaminofluorene/guanosine adduct, prepared by reaction of 2-acetoxy-2-acetylaminofluorene (2AAAF) with DNA, was a significant but unreliable marker of 2AAF/DNA adducts in the rat liver in vivo. DNA repair did not appear to remove DNA adducts selectively, and adducts remained in DNA when discernible DNA repair had ceased.