Furan-induced dose–response relationships for liver cytotoxicity,cell proliferation,and tumorigenicity (furan-induced liver tumorigenicity)

Rodent studies of furan are associated with liver cell necrosis, release of liver-associated enzymes, increased hepatocyte proliferation, and hepatocarcinogenesis. For carcinogens whose proposed mode of action is cytolethality, it is hypothesized that the dose–response curve for tumor development would parallel the dose–response curve for cell death with compensatory proliferation in the target organ. To prospectively test this hypothesis, female B6C3F₁ mice were exposed to furan at carcinogenic doses and lower for 3 weeks or 2 years. At 3 weeks and in the 2-year study, there were dose-dependent and significant increases in hepatic cytotoxicity at 1.0, 2.0, 4.0, and 8.0 mg furan/kg. For cell proliferation as measured by 5-bromo-2′-deoxyuridine (BrdU) labeling index (LI), there was a statistically significant trend with increasing dose levels of furan and increased LI at 8.0 mg/kg. There was an increased incidence of foci of altered hepatocytes, hepatocellular adenomas, and adenomas or carcinomas at 4.0 and 8.0 mg/kg and carcinomas at 8.0 mg/kg. The multiplicity of microscopic tumors was increased and latency was decreased in mice exposed to 8.0 mg/kg. Prevalence of hepatic nodules at necropsy was increased in mice exposed to 4.0 and 8.0 mg/kg. Data demonstrate an association among furan-induced hepatic cytotoxicity, compensatory cell replication, and liver tumor formation in mice; at high doses ?4.0 mg/kg, furan induced hepatotoxicity, compensatory cell replication and tumorigenesis in a dose-related manner, while furan did not produce tumors at cytotoxic doses of 1.0 and 2.0 mg/kg.     

Inter-laboratory comparison of turkey in ovo carcinogenicity assessment (IOCA) of hepatocarcinogens

In three independent laboratories carcinogens (diethylnitrosamine, DEN, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and non-carcinogens (N-nitrosoproline, nicotine) were evaluated in turkey eggs for in ovo carcinogenicity assessment (IOCA). Compounds were injected into aseptic fertilized eggs. After incubation for 24 days, foci of altered hepatocytes (FAH), some with a pseudoglandular structure and/or signs of compression of the surrounding tissue were observed in the fetal liver. All laboratories were able to distinguish unequivocally the hepatocarcinogen-exposed groups from those exposed to non-carcinogens or the vehicle controls, based on the pre-specified evaluation parameters: tumor-like lesions, pseudoglandular areas and FAH. In addition to focal changes, only the carcinogens induced hepatocellular karyomegaly. Lower doses of the carcinogens, which did not induce FAH, were sufficient to induce hepatocellular karyomegaly. After exposure to 4 mg DEN, gall bladder agenesis was observed in all fetuses. The IOCA may be a valuable tool for early investigative studies on carcinogenicity and since it does not use rodents may complement chronic rat or mouse bioassays. Test substances that are positive in both rodents and fertilized turkey eggs are most probably trans-species carcinogens with particular significance for humans. The good concordance observed among the three laboratories demonstrates that the IOCA is a reliable and robust method.     

Absence of chemopreventive influence of propolis on the rat liver altered foci development

Propolis (bee glue) is a complex mixture of natural substances that exhibits a broad spectrum of biological activities. As the possibility exists that it may exert a chemopreventive role against cancer development, the present study aimed to evaluate the chemopreventive influence of a Brazilian aqueous propolis extract (APE) in a rat two-stage (initiation-promotion) medium-term bioassay for chemical liver carcinogenesis. Male Wistar rats were sequentially initiated with diethylnitrosamine (DEN, 200 mg/kg b.w.) and, 2 weeks later, exposed to a diet containing hexachlorobenzene (HCB, 100 ppm) and to APE 0.1% through drinking water for 6 weeks. Appropriate control groups were also established. The animals were sacrificed at the weeks 8th and 30th when liver samples were processed to evaluate the development of altered hepatocyte foci (AHF) identified under hematoxylin and eosin (H&E) staining and by the immunohistochemical expression of the enzyme glutathione S-transferase placental form (GST-P). The results indicate that APE 0.1% did not protect against the development of any of the differentially identified putative preneoplastic foci in DEN-initiated animals, exposed or not to the promoting agent HCB. Also, APE 0.1% by itself did not significantly induce any AHF, what is in line with its already known absence of genotoxic potential. Our results indicate that an aqueous extract of Brazilian propolis did not exert chemoprevention on the hepatocarcinogenesis process chemically induced in the rat.     

Hepatocellular proliferation and hepatocarcinogen bioactivation in mice with diet-induced fatty liver and obesity

Human liver cancer is in part associated with obesity and related metabolic diseases. The present study was undertaken in a mouse model of diet-induced obesity (DIO) and hepatic steatosis, conditions which can be associated with hepatic neoplasia, to determine whether the rates of cell proliferation or hepatocarcinogen bioactivation were altered in ways which could facilitate hepatocarcinogenesis. DIO mice were generated by feeding C57BL/6 (B6) male mice a high-fat diet beginning at 4 weeks of age; age-matched conventional lean (LEAN) B6 mice fed a low fat diet (10% Kcal from fat) were used for comparison. Groups of 28 week old DIO and LEAN mice were dosed with the bioactivation-dependent DNA-reactive hepatocarcinogen 2-acetylaminofluorene (AAF), at 2.24 or 22.4 mg/kg, given by gavage 3 times per week for 31 days, or received no treatment (DIO and LEAN control groups). Compared with the LEAN control group, the DIO control group had a higher mean body weight (16.5 g), higher mean absolute (1.4 g) and mean relative (25.5%) liver weights, higher (394%) liver triglyceride concentrations, and an increased incidence and severity of hepatocellular steatosis at the end of the dosing phase. The DIO control group also had a higher mean hepatocellular replicating fraction (31% increase, determined by proliferating cell nuclear antigen immunohistochemistry). Hepatocarcinogen bioactivation, based on formation of AAF DNA adducts as measured by nucleotide ³²P-postlabeling, was similar in both DIO and LEAN AAF-dosed groups. Thus, hepatocellular proliferation, but not hepatocarcinogen bioactivation, was identified as an alteration in livers of DIO mice which could contribute to their susceptibility to hepatocarcinogenesis.     

Effect of vitamin C in reducing the toxicity of endosulfan in liver in rabbits

In this study, the effect of endosulfan, an organochlorine pesticide, and the ameliorating effect of vitamin C on the livers of New Zealand white rabbits were studied. Livers of the rabbits were examined grossly and histopathologically, and caspase-3 activity was detected by immunohistochemical methods. A total of twenty-four rabbits were divided into four groups (n=6). Rabbits in Group I (END) were daily given a sublethal dose of endosulfan (1 mg/kg bw) in corn oil by oral gavage for 6 weeks. Group II (END+C) received the same dose of endosulfan and additionally Vit C (20 mg/kg bw) every other day during this period. Group III (OIL+C) received corn oil daily by oral gavage and vitamin C every other day for 6 weeks. Group IV (OIL), the control group, received only corn oil daily, by oral gavage throughout the experiment. The concentration of α-endosulfan in the END group was higher in livers (0.102±0.012 ppb) than the β-endosulfan (0.072±0.001 ppb). Decreased accumulation of α and β endosulfan was observed in the END+C group (0.025±0.003 and 0.016±0.002 ppb, respectively) (p<0.0001). The most prominent gross findings at the necropsy were seen in the END group, in which swollen and pale livers were commonly observed. Hemorrhages, degenerations, necrosis, and in some rabbits bile duct hyperplasia were the marked histopathological findings of the END group. Caspase-3 positive reaction was more severe in this group than in the others. An ameliorating effect of Vit C on gross, histopathological and immunohistochemical findings was observed in the END+C group. The results revealed that endosulfan is highly toxic for rabbit livers. However, toxicity was decreased by Vit C treatment, which reduced the accumulation of endosulfan in livers four-fold.     

Can a single lower trunk body-fixed sensor differentiate between level-walking and stair descent and ascent in older adults? Preliminary findings

Stair ascent and descent are common forms of ambulation that may be challenging to detect. Here, we propose the first step towards differentiating between stair negotiation and level-walking using a single body-fixed sensor.Seventeen healthy older adults (age: 79.3 ± 4.2 years, 47% women) wore a body-fixed sensor on the lower-back while performing level-walking and stair negotiation. Measures derived from the 3D acceleration and angular-velocity signals included medians, ranges, step duration, step and stride regularity, filtered vertical to horizontal acceleration ratio (VAF/HAF), and wavelet-based features. Friedman's and Wilcoxon tests compared between conditions. Stepwise-binary logistic-regression evaluated classification accuracy.During level-walking, yaw range was lowest and anterior–posterior and vertical step and stride regularity were highest (p ≤ 0.007). Anterior–posterior step regularity (p = 0.003), VAF/HAF (p = 0.094), and yaw range (p = 0.105) identified level-walking (92.2% accuracy). During stair ascent, roll range, median anterior–posterior acceleration and anterior–posterior wavelet-coefficient were lowest (p ≤ 0.006), while VAF/HAF was highest (p = 0.0029). Anterior posterior wavelet coefficient (p = 0.038) and VAF/HAF (p = 0.018) identified stair ascent (94.3% accuracy). During stair descent, vertical and medio-lateral ranges were highest and medio-lateral stride regularity and VAF/HAF were lowest (p ≤ 0.006). VAF/HAF (p = 0.01), medio-lateral acceleration range (p = 0.069), and medio-lateral stride regularity (p = 0.072) identified stair descent (90.2% accuracy).These findings suggest that a single worn body-fixed sensor can be used to differentiate between level-walking and stair negotiation.     

Human hepatic preneoplasia: phenotypes and proliferation kinetics of foci and nodules of altered hepatocytes and their relationship to liver cell dysplasia

 Foci of altered hepatocytes (FAH) represent preneoplastic lesions, as shown in various animal models of hepatocarcinogenesis, but their significance in the human liver has not been established. The cellular composition, size distribution and proliferation kinetics of FAH in 163 explanted and resected human livers with or without hepatocellular carcinoma (HCC) and their possible association with small-cell change of hepatocytes (SCC) were therefore studied. FAH, including glycogen-storing foci (GSF), mixed cell foci (MCF) and basophilic cell foci, were found in 84 of 111 cirrhotic livers, demonstrating higher incidences in cases with (29/32) than in those without HCC (55/79). FAH were observed more frequently in HCC-free cirrhosis associated with hepatitis B or C virus or chronic alcoholic abuse (high-risk group) (37/47) than in that due to other causes (low-risk group) (12/21). MCF, predominant in cirrhotic livers of the high-risk group, were more proliferative, larger and more often involved in formation of nodules of altered hepatocytes (39.3%) than were GSF (8.5%). The results suggest that the FAH are preneoplastic lesions, MCF being more advanced than GSF. Oncocytic and amphophilic cell foci were also observed, but their significance remains to be clarified. Two types of SCC, namely diffuse and intrafocal SCC, were identified, but only intrafocal SCC was found to be related to increased proliferative activity and more frequent nodular transformation of the FAH involved, suggesting a close association with progression from FAH to HCC. Received: 11 March 1997 / Accepted: 2 June 1997     

Relevance of hepatic preneoplasia for human hepatocarcinogenesis

Different lesions have been suggested to represent preneoplastic conditions in human liver. They include liver cell dysplasia, separated in large-cell change (LCC) and small-cell change (SCC), adenomatoid hyperplasia, and the more recently identified foci of altered hepatocytes (FAH) and nodules of altered hepatocytes (NAH). FAH have been demonstrated to represent preneoplastic lesions in various animal models of hepatocarcinogenesis. To demonstrate prevalence and significance of FAH in the human liver, the cellular composition, size distribution, and proliferation kinetics of these lesions were studied in 163 explanted and resected human livers with or without hepatocellular carcinoma (HCC). FAH including glycogen-storing foci (GSF), mixed cell foci (MCF), and basophilic cell foci were found in 84 of 111 cirrhotic livers, demonstrating higher incidences in cases with than without HCC. MCF, predominant in cirrhotic livers of the high-risk group, were more proliferative, larger and more often involved in formation of NAH than GSF. The results suggest that the FAH are preneoplastic lesions, MCF being more advanced than GSP. We also investigated the relationship of FAH to liver cell dysplasia. Occurrence of SCC, rather than that of LCC, confers FAH an increased proliferation activity and higher risk to nodular transformation, and, hence, should be considered a precancerous condition. Histological detection of FAH and SCC through needle-aspiration liver biopsy can be used for monitoring HCC development in high-risk populations, such as HBV carriers with chronic hepatitis and/or cirrhosis.     

Effects of l-ascorbic acid on two cycles of cisplatin-induced DNA double-strand breaks and phosphorylation of p53 in the liver

Cisplatin, a commonly used anticancer drug, was studied to investigate its effects on structure, DNA damage and p53 along with the possible protective effects of l-ascorbic acid in the liver. Adult male BALB/c mice were treated with 0, 10 mg/kg l-ascorbic acid and two cycles of cisplatin 1 mg/kg/2.5 mg/kg with or without l-ascorbic acid (17 days recovery period between the cycles) and the livers were collected at 72 h after the last exposure. Structural damage was analyzed in Masson's trichrome and Hortega's silver stained liver tissues. The DNA double-strand breaks with duplex 3′ overhangs and 5′ P-blunt ends were labeled by in situ oligo ligation by using hairpin oligonucleotide probes. The expression of p53 and phosphorylated p53 (p-p53) was detected by immunohistochemistry. Structural changes such as vacuolization of hepatocytes, pyknosis, infiltration of leukocytes and pericentral fibrosis were observed without any protection from l-ascorbic acid. The reticular fibrous framework was affected and the incidence of Kupffer cells was decreased. Cisplatin induced the DNA double-strand breaks (p < 0.001); however, the latter appeared in a p53-independent, but p-p53-dependent manner. l-ascorbic acid showed significant protective effect on cisplatin-induced DNA damage (p < 0.001). Cisplatin also enhanced p53 phosphorylation in a dose-dependent manner and l-ascorbic acid reduced this biochemical change only in 1 mg/kg group. In conclusion, cisplatin-induced structural changes are not, but the DNA damage and phosphorylation of p53 are, significantly, but not completely, alleviated by l-ascorbic acid.     

Histopathological findings in livers of patients with urea cycle disorders

BackgroundUrea cycle disorders (UCD) are caused by genetic defects in enzymes that constitute the hepatic ammonia detoxification pathway. Patients may present with variable clinical manifestations and with hyperammonaemia. Liver abnormalities have been associated with UCD, but only a few reports on the histopathological findings in the liver of UCD patients have been published.MethodsWe conducted a retrospective review of liver biopsies, ex-planted livers and livers at post-mortem of patients with UCD. A single pathologist reviewed all specimens.ResultsThere were 18 liver samples from 13 patients with confirmed UCD: four ex-planted livers from patients with Ornithine Transcarbamylase (OTC) (n = 3) and Carbamoyl Phosphate Synthetase 1 (CPS 1) (n = 1) deficiencies, eight post-mortem samples from patients with CPS 1 (n = 2), OTC (n = 4), Argininosuccinate Synthetase (ASS) (n = 1) and Argininosuccinate Lyase (ASL) (n = 1) deficiencies, and six liver biopsies, three of which came from one patient with ASL deficiency. The other three liver biopsies were from patients who subsequently received liver transplantation. Histopathological findings in samples from neonates were non-specific. Samples from three late onset OTC deficient and one ASL deficient patients showed thin fibrous septa with portal to portal bridging fibrosis and focal marked enlargement and pallor of the hepatocytes due to accumulation of glycogen particles, resembling glycogenosis and resulting in a prominent nodular pattern. Serial liver biopsies in four UCD patients with interval between samples ranging from 1 year 2 months to 17 years showed progression in fibrosis in one OTC and one ASL deficient patients. Moderate fatty changes to no progression in liver disease were noted in the two patients (OTC = 1 and CPS = 1). A variety of non-specific features such as fatty change, mild inflammation, cholestasis and focal necrosis were seen in the other UCD patients.ConclusionsHistopathological changes in livers from neonates with UCD are non-specific. Older patients with UCD seem to show variable hepatic fibrosis compared to those who died early. Some of these patients also show focal and superficial resemblance to a glycogen storage disorder and cirrhosis. However, progression of these changes seems to be slow. To clarify the long term consequence of these changes, more extensive periods of follow up in a larger population series is needed.     

Overexpression of insulin receptor substrate-1 emerges early in hepatocarcinogenesis and elicits preneoplastic hepatic glycogenosis.

Insulin receptor substrate-1 (IRS-1) is a multisite docking protein occupying a central position in signaling cascades stimulated by a number of growth factors including insulin. Using Western blotting and immunohistochemistry, we investigated the expression of IRS-1 in more than 400 preneoplastic foci of altered hepatocytes and in 12 hepatocellular carcinomas induced in rats by oral administration of N-nitrosomorpholine. In both N-nitrosomorpholine-treated and untreated rat livers, IRS-1 was demonstrable by Western blotting, but with the exception of a few single hepatocytes it was not detectable in the normal parenchyma by immunohistochemistry. In contrast, immunohistochemistry revealed that IRS-1 was strongly expressed in the majority of foci of altered hepatocytes particularly in approximately 97% of the clear/acidophilic and mixed cell foci showing excessive storage of glycogen (glycogenosis). In glycogen-poor basophilic foci of altered hepatocytes and hepatocellular carcinomas, IRS-1 was not detected by immunohistochemistry, but a weak expression was observed in small subpopulations of three hepatocellular carcinomas containing remnants of glycogen. These results indicate that the focal overexpression of IRS-1 is an early event in hepatocarcinogenesis, which is closely correlated with preneoplastic hepatic glycogenosis. During progression from glycogenotic foci to hepatocellular carcinomas, IRS-1-overexpression is gradually down-regulated, and this late event is associated with a fundamental metabolic shift leading to the malignant neoplastic phenotype.     

Biochemical and histopathological evaluation of histamine receptors (H1R,H2R,H3R and H4R)-agonist in rabbits

The present study was designed to investigate the biochemical and histopathological changes in the livers of rabbits treated with histamine and histamine receptors (H1R–H4R)-agonist. The cohort comprised of six groups containing five rabbits each. Control group received sterile distilled water (1 mL/kg × b.i.d.) and treated groups received subcutaneous histamine (100 μg/kg × b.i.d.) and H1R–H4R-agonist (histamine trifluoro-methyl toluidide, amthamine, R-[?]-α-methylhistamine, clobenpropit, respectively) each in a dose of 10 μg/kg × b.i.d. (12 h [8 am and 8 pm]) for 30 days. Hepatotoxicity due to these agonists was analyzed using biochemical and histopathological methods. Histamine and H1R–H3R-agonist were found to be hepatotoxic as shown by statistically significant (p < 0.05) elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), most marked in the H3R-agonist group. However, their levels in H4R-agonist group remained very similar to the control group. The entire drug treated groups as compared to control showed significant elevated levels of alkaline phosphatase (ALP). Histopathological examination revealed obvious changes in histamine, H2R- and H3R-agonist groups in terms of alteration of hepatic microstructure, congestion, focal necrosis and increased incidence of multinucleate hepatocytes while H1R and H4R groups showed minimal changes. From the findings of the present study it may be concluded that on repeated administration, histamine and HR-agonists-induced hepatotoxicity which is most pronounced with H3R-agonist though not severe enough to jeopardize the vital functions of liver and warrants further long-term studies.     

Transient disruption of liver gap junctional intercellular communication and induction of apoptosis after administration of 1,4-bis[2-(3,5 dichloropyridyloxy)]benzene in mice

Gap junctional intercellular communication (GJIC) and connexin expression (Cx26 and Cx32) in mouse liver were studied after administration of 4-bis[2-(3,5 dichloropyridyloxy)]benzene (TCPOBOP), a phenobarbital-like enzyme inducer. Female C57Bl/6 mice were administered TCPOBOP (5.8 mg/kg BW) and euthanized 0, 24, 48 and 72 hours later. Liver samples were snap frozen, or fixed in formalin, or submitted to GJIC analysis. The proliferating cell nuclear antigen (PCNA) immunohistochemistry and the Western blotting for Cx26 and Cx32 were performed. After 48 and 72 h of drug administration the liver-to-body weight ratio was increased 70% and 117% (p<0.0001), respectively. There were temporal-dependent alterations in liver histopathology and a significant increase in cell proliferation was noted after 48 h and sustained after 72 h, though to a lesser extent (p<0.0001). In addition, TCPOBOP administration induced apoptosis, which appeared to be time-dependent showing statistical significance only after 72 h (p<0.0001). Interestingly, a transient disruption by nearly 50% of GJIC capacity was detected after 48 h of drug ingestion, which recovered after 72 h (p=0.003). These GJIC changes were due to altered levels of Cx26 and Cx32 in the livers of TCPOBOP-treated mice. We concluded that a single administration of TCPOBOP transiently disrupted the levels of GJIC due to decreased expression of connexins and increased apoptotic cell death in mouse liver.     

Antidiabetic,antihyperlipidemic and antioxidant effects of the flavonoid rich fraction of Pilea microphylla (L.) in high fat diet/streptozotocin-induced diabetes in mice

The present study describes the antidiabetic effect of the flavonoid rich fraction of Pilea microphylla (PM1). HPLC characterization of PM1 revealed the presence of polyphenols viz., chlorogenic acid, rutin, luteolin-7-O-glucoside, isorhoifolin, apigenin-7-O-glucoside, and quercetin. PM1 inhibited dipeptidyl peptidase IV (DPP-IV) in vitro with an IC₅₀ of 520.4 ± 15.4 μg/ml. PM1, at doses of 300, 600 and 900 mg/kg i.p., also produced dose-dependent mean percent reductions of 9.9, 30.6 and 41.0 in glucose excursion (AUC_0–120 min) respectively in lean mice. However, even the highest dose of PM1 did not alter normoglycemic condition. PM1 at dose of 100 mg/kg/day, i.p. for 28 days produced significant (p < 0.05) reduction in body weight, plasma glucose (PG), triglycerides (TG) and total cholesterol (TC) content in high-fat streptozotocin-induced diabetic mice. PM1 also improved oral glucose tolerance significantly (p < 0.05) with mean percentage reduction of 48.0% in glucose excursion (AUC_0–120 min) and significantly (p < 0.05) enhanced the endogenous antioxidant status in mice liver compared to diabetic control. PM1 preserved islet architecture and prevented hypertrophy of hepatocytes as evident from the histopathology of pancreas and liver. PM1 did not show any detectable hematological toxicity at therapeutic doses. In conclusion, PM1 exhibits antidiabetic effect possibly by inhibiting DPP-IV and improving antioxidant levels in high fat diet/streptozotocin (HFD/STZ) diabetic mice.     

Dose-dependent hepatoprotective effect of emodin against acetaminophen-induced acute damage in rats

Protective effect of emodin (1,3,8-trihydroxy-6-methyl anthraquinone), an active compound of Ventilago madraspatana Gaertn., was evaluated against acetaminophen-induced biochemical and histological alterations in rats. Acetaminophen (2 g/kg, po) administration caused significant elevation in the release of serum transaminases, alkaline phosphatase, lactate dehydrogenase, serum bilirubin and serum protein with concomitant decrease in hemoglobin and blood sugar after 24 h of its administration. Toxicant exposure intensified the lipid peroxidation and altered glutathione status, activities of adenosine triphosphatase, acid phosphatase, alkaline phosphatase as well as major cellular constituents i.e., protein, glycogen and total cholesterol in liver and kidney. Treatment of emodin (20, 30 and 40 mg/kg, po) significantly lessened the toxicity by protecting acetaminophen-induced alterations in various blood and tissue biochemical variables after 24 h of its administration. Acetaminophen administration initiated histological damage in liver. Some degree of protection was seen after emodin therapy in a dose-dependent manner. Emodin at doses of 30 and 40 mg/kg effectively reversed toxic events induced by acetaminophen as same as silymarin (50 mg/kg, po). Thus, the study concluded that emodin at a dose of 30 mg/kg (po) possesses optimum hepatoprotective ability against acetaminophen-induced toxicity.     

Lack of promoting effects from physical pulmonary collapse in a female A/J mouse lung tumor initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with remarkable mesothelial cell reactions in the thoracic cavity by the polymer

Experimental identification of potential chemopreventive or tumor promotive agents in the lung is important. Establishment of short-term bioassay models is therefore a high priority. In an attempt to induce strong promotion effects, in Experiment 1, left thoracotomy was performed on A/J mice at week 3 after initiation with 4-(methylnitrosamno)-1-(3-pyridyl)-1-butanone (NNK) (2 mg/0.1 ml saline/mouse i.p.) at weeks 0 and 1. In Experiment 2, at week 3, 0.2 ml of polymer gel was infused directly into the left cavity of the thorax with thoracotomy to occupy certain thoracic cavity volume and to examine the influence of physical pulmonary collapse. The experiments were terminated after 8, 10, 12 and 16 weeks in Experiment 1, and 12 weeks in Experiment 2 but no clear promotion effects in either experiment or pulmonary collapse due to infused polymer were apparent. However, a pronounced mesothelial cell reaction to the infused polymer was evident on the left lung surfaces and parietal pleura in Experiment 2. In conclusion, the present experiments did not demonstrate any clear lung tumor promotion effects of thoracotomy or physical left lung collapse. It remains possible, however, that alternative approaches might have greater efficacy and these need more consideration. In addition, mesothelial cells reaction was observed with the infused polymer.     

Preliminary evaluation of in vitro cytotoxicity and in vivo antitumor activity of Premna herbacea Roxb. in Ehrlich ascites carcinoma model and Dalton's lymphoma ascites model

In the present study, the root nodules of Premna herbacea Roxb. (PH) was investigated for its in vitro cytotoxicity and in vivo antitumor activity. Two extracts, aqueous and alcoholic; two fractions of alcoholic extract, ethyl acetate and butanol fractions were screened for their in vitro cytotoxicity by brine shrimp lethality (BSL) assay, trypan blue exclusion assay and MTT assay. Alcoholic extract and its ethyl acetate fraction were found to be the most effective in BSL assay, trypan blue exclusion assay. In vivo antitumor activity was screened in the Ehrlich ascites carcinoma (EAC) model and the Dalton lymphoma ascites (DLA) model. The extracts and the fractions were tested at two dosages (250 and 500 mg/kg) by intraperitoneally (i.p.) route on every alternate day upto 13th day. Cisplatin was used as positive control in both studies in single dose (day 1) 3.5 mg/kg by i.p. route. In EAC model, ascites tumor was induced by inoculating 2.5 million of EAC cells i.p. alcoholic extract at 500 mg/kg was the most effective in elevating MST, reduction in body weight in EAC induced tumor. Only the effective extract i.e., alcoholic extract were studied for hematological and antioxidant parameter. It showed a restoring effect on altered hematological parameters and a significant improvement in biochemical parameters at 250 mg/kg dose of alcoholic extract. These results explain the toxicity of 500 mg/kg might be high. In the Dalton lymphoma ascites (DLA) model, solid tumor was developed by i.m. injection of 1 million DLA cells. Both the extracts and the fractions possessed potent antitumor activity against solid tumor models by significantly reducing the solid tumor weight and volume.     

Low Serum Levels of Total Rabbit-IgG Is Associated with Acute Graft-Versus-Host Disease after Unrelated Donor Hematopoietic Stem Cell Transplantation: Results from a Prospective Study

In a prospective study we determined rabbit-IgG (r-IgG) levels in serum samples before (day 0) and after (days 1 and 7) unrelated donor hematopoietic stem cell transplantation (HSCT). Most patients suffered from a hematologic malignancy. All patients received rabbit antithymocyte globulin (ATG) as part of the conditioning for 2-4 days (2 mg/kg/day). We found a good correlation (r = 0.34, r = 0.42 and r = 0.46) between the dose of ATG and serum r-IgG levels at all 3 time points. The cumulative incidence of acute graft-versus-host disease (aGVHD) grades II-IV in patients given the 4, 6, and 8 mg/kg ATG dose was 50%, 34%, and 15% (P = .04), respectively. In patients with r-IgG ≤70 μg/mL (n = 54) the cumulative incidence of grades II-IV aGVHD was 33% compared with only 6% in those with r-IgG >70 μg/mL (n = 18), P = .023. Low serum levels of r-IgG seem to be a strong predictor for aGVHD grades II-IV in patients treated with Thymoglobulin before unrelated donor HSCT.     

Co-supplementation of single and multi doses of vitamins C and E ameliorates cisplatin-induced acute renal failure in mice

Higher doses of antioxidant vitamins C and E have been proved to be effective against cisplatin-induced nephrotoxicity in animals. However, the possible effective equivalent dose in human was found to be higher than that of the upper tolerable intake level (UL) for these vitamins. Hence, the current study was aimed to evaluate the protective effect of co-supplementation of single and multi doses of vitamins C and E against cisplatin-induced acute renal failure in mice. Single dose of vitamin C (500 mg/kg), vitamin E (500 mg/kg), and vitamin C plus vitamin E (250 mg/kg each) were administered orally 1 h prior to cisplatin (12 mg/kg, i.p) injection, whereas in a multidose study they were administered 1 h prior, and 24 and 48 h after the cisplatin injection. Serum urea and creatinine levels were estimated 72 h after the injection of cisplatin. Renal concentrations of glutathione (GSH) and malondialdehyde (MDA) were also determined. Co-supplementation of vitamins significantly protected the cisplatin-induced increased levels of serum urea, creatinine, renal MDA, and the declined renal GSH level. Administration of single and multi doses of vitamin C plus E (250 mg/kg each) rendered significant nephroprotection. Therefore, accounting for the rare side effect from high intake of vitamins C and E observation of this study indicates that a multidose combination therapy of these vitamins at their lower doses can be effective in protecting the cisplatin-induced renal damage. The protection is partially mediated through the antioxidant effect of the vitamins.