Effect of DNA synthesis on induction of preneoplastic and neoplastic lesions in rat liver by a single dose of methylazoxymethanol acetate

A single intravenous injection of methylazoxymethanol (MAM) acetate in doses of either 20 or 35 mg/kg body weight to male Sprague-Dawley rats induced altered liver cell foci and later, liver neoplasms in a dose related manner. Sequential observations in the rats given 35 mg/kg and thereafter fed an iron-loading diet revealed that the number of iron-excluding foci/cm2 increased with time. Partial hepatectomy (PH) before the high dose of MAM acetate resulted in 100% lethality while hepatectomy before the low dose carcinogen exposure lead to a higher incidence of neoplasms than in rats that received carcinogen alone. PH after either high or low dose carcinogen exposure did not result in a greater occurrence of liver neoplasms.     

ESP-102, a standardized combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, significantly improved scopolamine-induced memory impairment in mice

We assessed the effects of oral treatments of ESP-102, a standardized combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, on learning and memory deficit. The cognition-enhancing effect of ESP-102 was investigated in scopolamine-induced (1 mg/kg body weight, s.c.) amnesic mice with both passive avoidance and Morris water maze performance tests. Acute oral treatment (single administration prior to scopolamine treatment) of mice with ESP-102 (doses in the range of 10 to 100 mg/kg body weight) significantly reduced scopolamine-induced memory deficits in the passive avoidance performance test. Another noteworthy result included the fact that prolonged oral daily treatments of mice with much lower amounts of ESP-102 (1 and 10 mg/kg body weight) for ten days reversed scopolamine-induced memory deficits. In the Morris water maze performance test, both acute and prolonged oral treatments with ESP-102 (single administration of 100 mg/kg body weight or prolonged daily administration of 1 and 10 mg/kg body weight for ten days, respectively, significantly ameliorated scopolamine-induced memory deficits as indicated by the formation of long-term and/or short-term spatial memory. In addition, we investigated the effects of ESP-102 on neurotoxicity induced by amyloid-beta peptide (Abeta25-35) or glutamate in primary cultured cortical neurons of rats. Pretreatment of cultures with ESP-102 (0.001, 0.01 and 0.1 mug/ml) significantly protected neurons from neurotoxicity induced by either glutamate or Abeta25-35. These results suggest that ESP-102 may have some protective characteristics against neuronal cell death and cognitive impairments often observed in Alzheimer's disease, stroke, ischemic injury and other neurodegenerative diseases.     

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.     

Dehydroepiandrosterone (DHEA) facilitates liver regeneration after partial hepatectomy in rats

In this study we investigated whether or not liver regeneration is facilitated by dehydroepiandrosterone (DHEA) after partial (70%) hepatectomy in rats. Treatment with DHEA (300 mg/kg body weight) did not cause any significant increase in the expression ratio of proliferating cell nuclear antigen (PCNA) in sham-operated controls; however, in partially hepatectomized rats it caused a significant increase in the ratio in hepatocytes 24 and 36 hr after hepatectomy. In partially hepatectomized rats, DHEA treatment significantly accelerated the restoration of liver 48, 60, and 72 hr after partial hepatectomy. The restoration rate in DHEA-treated hepatectomized rats at 72 hr was 1.3-fold greater than in partially hepatectomized controls. Treatment with androstenedione (300 mg/kg body weight), the first metabolite of DHEA, did not cause any significant increase in the expression of PCNA in either sham-operated controls or partially hepatectomized rats. These results indicate that DHEA itself promotes the liver regenerative process after partial hepatectomy in rats.     

Cytogenetic effects of pesticides. III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone

The production of micronuclei in mouse bone marrow by the pyrethroid insecticide, cypermethrin and the botanical insecticide, rotenone was examined. Three routes of administration were used for the insecticides: intraperitoneal, oral and dermal. The different routes of treatment with cypermethrin and rotenone caused toxicity of marrow as indicated by a significant increase in the percentage of polychromatic erythrocytes (PEs) over that of the control. Cypermethrin showed mutagenic potential as evidenced by a positive response in the micronucleus assay. Oral administration of the insecticide at a dietary level of 900 ppm for 7 and 14 consecutive days as well as double and multiple (total 4) dermal treatments (360 mg/kg body wt.) induced a statistically significant increase in the frequency of PEs with micronuclei. The conducted intraperitoneal (i.p.) treatments with cypermethrin: single injection at 60 and 180 mg/kg body wt., double and multiple injections (total 3) at 60 mg/kg body wt. did not affect the percentage of PEs with micronuclei. The different treatments with rotenone: single, double and multiple (i.p.) injections (total 3) at 2 and 3 mg/kg body wt., oral administration for 14 consecutive days at dietary level of 225 ppm and multiple dermal treatments (total 4) with 135 mg/kg body wt. showed no effect on the frequency of micronuclei in PEs.     

Enhancement of aflatoxin B1-induced hepatocarcinogenesis in rats by partial hepatectomy

The effect of enhanced cell replication induced by partial hepatectomy (PH) in aflatoxin B1 (AFB1)-induced hepatocarcinogenesis has been studied in rats of the inbred As2 strain. Animals were given 0.25 mg/kg body weight of AFB1 as a single intraperitoneal dose 24 h after PH. Non-hepatectomized animals given the same dose of AFB1 served as controls. Neoplastic nodules and hepatocellular carcinoma (HCC) were detected respectively in 100% and 90% of hepatectomized animals sacrificed between 55 and 65 weeks after AFB1 administration. None of the ten non-hepatectomized rats sacrificed at this time interval showed HCC or neoplastic nodules. On histochemical staining the tumour population was found to be heterogeneous. Thus PH resulted in enhancement of AFB1-induced hepatocarcinogenesis in rats of the AS2 strain.     

Liposome-encapsulated oxymorphone hydrochloride provides prolonged relief of postsurgical visceral pain in rats

Adequate pain control is necessary for optimal postsurgical recovery and humane treatment of laboratory and companion animals. Opioid drugs are currently the most potent analgesic agents available in human and veterinary medicine. Long-acting formulations of opioid drugs confer several important advantages over standard pharmaceutical preparations, especially for use in animals. A long-acting formulation of oxymorphone hydrochloride was produced by encapsulation into liposomes. Liposome-encapsulated (LE) oxymorphone was tested in a rat model of visceral postoperative pain. Rats were given one subcutaneous injection of LE oxymorphone (1.2 or 1.6 mg/kg of body weight) or standard oxymorphone (0.3 mg/kg) at the time of intestinal transection or resection. A single administration of LE oxymorphone hydrochloride was as effective for relief of postoperative pain in rats (P = 0.18), as were multiple (q4 h or q8 h) injections of 0.3 mg/kg of the standard pharmaceutical preparation. The rats given LE oxymorphone prior to intestinal resection also had significantly higher body weight at three and seven days after surgery than did rats that were given standard oxymorphone. In conclusion, LE oxymorphone was effective in treating visceral pain associated with intestinal surgery in rats. On the basis of body weight gain, rats treated with LE oxymorphone had improved recovery outcome, compared with rats treated with repeated injections of standard oxymorphone.     

Leishmania donovani: Therapeutic and prophylactic action of antimony dextran glycoside (RL-712) in the golden hamster

Antimony dextran glycoside (RL-712, manufactured by Rosco A/S Pharmaceutical Taastrup, Denmark) produced a remarkable decrease in Leishmania donovani densities in hamster spleen and liver when administered in a single intraperitoneal (ip) dose of 500 mg Sb/kg body weight or in 600 mg Sb/kg body weight divided into two or three equal parts injected at weekly intervals beginning 15 days after intracardial inoculation of 10 million amastigotes, and also in a single ip dose of 300 mg Sb/kg body weight 14, 7, and 0 days before intracardial inoculation of 10 million amastigotes. No parasites were detected on using a single dose of 500 mg Sb/kg body weight followed by 100 mg Sb/kg body weight one week later.When the drug was administered in a dose of 500 mg Sb/kg body weight either 10 days before subcutaneous inoculation of 60 million promastigotes or eight days before intracardial inoculation of five million promastigotes, no parasites were recovered by smear or culture from the spleen and liver.Results show the prophylactic and therapeutic actions of this drug against L. donovani in the hamster.     

Modification of cyclophosphamide-induced clastogenesis and apoptosis in rats by alpha-lipoic acid

The aim of the present study was to investigate the protective efficacy of alpha-lipoic acid (LA) on the cyclophosphamide (CP)-induced chromosomal aberrations (CA) and apoptosis in the bone marrow of rats. Male Wistar rats of 140+/-20 g were categorized into eight groups. Five groups were administered CP (40 mg/kg body weight, intraperitoneally) to induce toxicity; four of these groups received a single intraperitoneal injection of LA at a dose of either 100 or 200 mg/kg body weight, and either 30 or 60 min prior to CP administration. A vehicle-treated control group and LA control groups were also included. Twenty-four hours after CP treatment, the frequency of CA in bone marrow cells were significantly increased in comparison with the controls. The CP-induced CA were associated with significant increase in DNA damage in the bone marrow as evidenced by increased single strand breaks, whereas in rats treated with LA and CP, the frequency of CA and single strand breaks were significantly decreased in comparison to those given CP alone. CP administration distinctly triggered the apoptotic and necrotic cell death, and LA pretreatment affected cell death by decreasing the number of apoptotic and necrotic cells. The protective effect of LA was found to be stronger at a dose of 200 mg/kg body weight than 100 mg/kg body weight dosage, indicating the dose dependent protective effect of LA. However, the protection by LA was not dependent on the time intervals between LA and CP administration. The results of this study illustrate the protective effect of LA on the CA and apoptosis induced by CP in the erythropoietic system of rats.     

"In vivo" (35S)-methionine interaction with rat-liver DNA and the effect of chemical carcinogens

1. After intraperitoneal administration of (35S)methionine (25 mg, 1.6 mCi/kg), detectable amount of radioactivity resulted associated to rat-liver DNA: the interaction reached the maximum value (about 18 pmol/mumol DNA P) by 2 h after administration of radioactive aminoacid. 2. The (35S)-binding was inhibited by the hepatocarcinogenic ethionine and dimethylnitrosamine, and was stimulated by the non-hepatocarcinogenic methylnitrosourea. 3. Hplc analysis of (35S)DNA enzymic digest evidenced two radioactive compounds, the UV behaviour of which is reported.     

STUDIES ON LIVER REGENERATION: II. EFFECTS of PHENOBARBITAL ON the ONSET and PATTERN of RAT LIVER REGENERATION FOLLOWING PARTIAL HEPATECTOMY

Abstract. Partially hepatectomized rats were given a single intraperitoneal injection of a phenobarbital solution or of water immediately after surgery. At various time intervals following the operation, the animals were injected with ¹³¹ iododeoxyuridine (¹¹³IDU), sacrificed 2 hr later, and radioactivity retained in formalin-fixed liver tissue was determined as a measure of DNA synthesis at the time of administration of the labeled precursor. In control animals without phenobarbital treatment, ¹³¹IDU incorporation into liver began to increase between 14 and 16 hr after partial hepatectomy. Phenobarbital treatment (0.1 mg per g of body weight) resulted in a delay of the increase in ¹³¹IDU incorporation by several hours. This delay was observed in animals subjected to partial hepatectomy in the morning as well as in those operated on in the evening. After phenobarbital treatment, the increase of mitotic activity was either delayed or occurred more slowly. the results are compared with the reported effects of partial hepatectomy on the time course of microsomal enzyme induction by phenobarbital.     

Protective effect of hesperidin on nicotine induced toxicity in rats

Nicotine administration (2.5 mg/kg of body weight, sc, 5 days a week for 22 weeks) enhanced lipid peroxidative indices (thiobarbituric acid reactive substances and hydroperoxides) accompanied by a significant increase in the marker enzymes alanine transaminase, aspartate transaminase, alkaline phosphatase and lactate dehydrogenase and elevated levels of cholesterol, triglycerides, phospholipids and free fatty acids in Wistar rats. There was a significant protection by hesperidin administration at different doses (25, 50, 75, 100 and 150 mg/kg body weight) in nicotine-treated rats. However, the effect of hesperidin was more significant at 25mg/kg dose. The results suggest that hesperidin exerts the protective effects by modulating the extent of lipid peroxidation. The results are supported by histopathological observations of lung, liver and kidney.     

The induction of latent liver damage by cyclophosphamide

The effect of the cyclophosphamide on the changes of the mitotic index (MI), metaphase-prophase ratio (M/P) and chromosomal aberrations in postmetaphase in regenerating rat liver was studied. Cyclophosphamide was injected in the single doses of 100 and 200 mg/kg i.p. 2 hours before partial hepatectomy (PHE). The results have shown that cyclophosphamide caused latent liver damage which was manifested after PHE in reduced MI and increased frequency of chromosomal aberrations. The extent of the changes induced by cyclophosphamide corresponds to that due to the effect of 3 or 6 Gy of X-rays, respectively.     

DEN+2-AAF-induced multistep hepatotumorigenesis in Wistar rats: supportive evidence and insights

Diethylnitrosamine (DEN), found in many commonly consumed foods, has been reported to induce cancers in animals and humans. Several models have been developed to study multistage carcinogenesis in rat liver; these include the Solt–Farber-resistant hepatocyte model. In the Solt–Farber model, the initiation consists of either a necrogenic dose of a hepatocarcinogen or a non-necrogenic dose in conjunction with partial hepatectomy (PH). We report a novel protocol for tumor induction in liver which eliminates the need for PH. Male Wistar rats were injected with single i.p. dose of DEN (200 mg/kg body weight), controls received saline only. After 1 week of recovery, the DEN-treated animals were administered with the repeated doses of 2-acetyamino fluorine (150 mg/kg body weight) orally in 1 % carboxymethyl cellulose that served as promoting agent. Thirty days after the DEN administration, hepatocellular damage was observed as evident by histopathological analysis. The marker enzyme analysis showed elevated levels of serum AST, ALT, and alkaline phosphatase and a decrease in the levels of liver superoxide dismutase and catalase. The oxidative stress in liver was confirmed by elevated levels of lipid peroxidation and a decrease in antioxidant parameters.     

The effect of single and of multiple doses of prednisolone tertiary butyl acetate on cell population kinetics in the small bowel mucosa of the rat.

The effect of single and of multiple doses of prednisolone upon cell population kinetics in the rat jejunal crypt was investigated, using autoradiography and stathmokinetic techniques with vincristine. Single injections of prednisolone (2.5 mg/kg body weight) induced a depression both flash thymidine labelling and mitotic indices; this change was shown to be due to a decreased cell production rate. Recovery of these proliferative indices occurred over seven days after injection; measurement of crypt size parameters showed a transient decrease in crypt population. Multiple daily injections of prednisolone (1 mg/kg body weight) produced a more sustained decrease in labelling and mitotic indices, which lasted as long as injections were continued (7 days); stathmokinetic techniques showed decreases in cell production rates, and the crypt population was also depressed throughout this period. It is concluded that prednisolone depresses cell proliferative rates in rat jejunal mucosa.     

Evidence for suppression of serum LH without elevation in serum estradiol or prolactin with a brain-enhanced redox delivery system for estradiol

We developed a redox system for brain-enhanced delivery of estradiol based on an interconvertible dihydropyridine in equilibrium pyridinium salt carrier. Estradiol (E2), when combined with the lipoidal carrier, readily crosses the blood-brain barrier. The carrier, when oxidized, reduces the rate of exit of the estradiol-carrier complex from the brain. Subsequent hydrolysis of the carrier provides sustained production of estradiol in the brain. The aim of the study was to evaluate the effects of single vs. multiple injections of the estradiol-chemical delivery system (E2-CDS) on both central and peripheral estrogen-responsive tissues. Ovariectomized Sprague-Dawley rats received an intravenous injection of E2-CDS at 10, 33, 100 or 333 micrograms/kg BW or the drug vehicle, dimethyl sulfoxide (DMSO; 0.5 ml/kg) every 2 days for 7 injections (2 weeks) or a single injection only at 2 days before sacrifice. With a single injection, E2-CDS did not affect serum luteinizing hormone (LH) levels at the 10 micrograms/kg dose but caused a dose-dependent reduction in serum LH of 39-52% at the dose range of 33 to 333 micrograms/kg. By contrast, multiple injections of E2-CDS caused a 32 to 76% reduction in serum LH levels at doses ranging from 10 micrograms/kg to 333 micrograms/kg. Additionally, multiple doses of E2-CDs caused a dose-dependent reduction in body weight at the 10 and 33 micrograms/kg doses with the higher doses causing no further weight reduction. For both single and multiple dosage groups, serum E2 levels remained unchanged after doses of E2-CDS of 10 and 33 micrograms/kg, then increased to 21 pg/ml for the single dosage group and to 23 pg/ml for the multiple dosage group at the 100 micrograms/kg dose, and to 59 pg/ml for singly-injected rats and 60 pg/ml for multiply-injected rats at the 333 micrograms/kg dose. Serum prolactin concentrations were closely correlated with serum E2 levels for both the single and multiple dose groups. These data reveal that a single or multiple doses of E2-CDS can reduce serum LH levels without elevating serum E2 or prolactin concentrations, supporting the concept of brain-enhanced delivery of estradiol with an estradiol chemical delivery system.     

Effects of clenbuterol and propranolol on muscle mass. Evidence that clenbuterol stimulates muscle beta-adrenoceptors to induce hypertrophy.

1. A single subcutaneous injection of clenbuterol hydrochloride (0.125 mg/kg body wt.) to female Wistar rats produced a rapid increase in muscle cyclic AMP and lactate concentrations and a decrease in muscle glycogen concentrations. These changes are characteristic of muscle beta-adrenoceptor stimulation and were abolished by intraperitoneal injection of propranolol (12.5 mg/kg) 15 min before clenbuterol administration. 2. When this dose of clenbuterol was injected twice daily, the changes in muscle metabolite concentrations which followed its acute administration persisted until day 7 of treatment, and were accompanied by increases in muscle mass, body weight and muscle protein synthesis rate (ks). When the clenbuterol injections were preceded by propranolol injections (12.5 mg/kg administered according to the protocol described above), or if animals were treated with propranolol only, the values of these variables were not significantly different from those of sham-injected controls. 3. In rats fed on a semi-synthetic diet (PW3) supplemented with 2 mg of clenbuterol/kg of diet for 7 days, the muscle mass was greater than that of rats fed on unsupplemented PW3. The increased muscle mass was accompanied by increased muscle lactate and decreased muscle glycogen concentrations. When PW3 was supplemented with 2 mg of clenbuterol/kg and 200 mg of propranolol/kg, the increase in muscle mass remained, but decreased muscle glycogen concentrations and increased muscle lactate concentrations were also observed. 4. These data are consistent with the hypothesis that clenbuterol influences muscle growth via beta-adrenoceptor stimulation.     

Effect of a beta-adrenergic antagonist on blood pressure, heart rate and beta-adrenoceptors in turkey poults

An irreversible beta-adrenergic blocker, bromoacetylalprenololmenthane (BAAM), was administered both peripherally and centrally to turkey poults, Meleagris gallopavo. Peripheral administration of BAAM (60 mg/kg body weight) effected a significant reduction in blood pressure and heart rate. Twenty minutes postinjection, mean blood pressure and heart rate were reduced 34.5 and 24.2%, respectively. Two days later, mean blood pressure values remained significantly depressed at 17.3% below preinjection determinations. Biochemical analysis of heart tissue following peripheral (intraperitoneal) injections of BAAM (60 mg/kg body weight) showed a significant decrease in beta-adrenergic receptors (BAR). Little or no change in the number of BAR in brain tissue was observed. Central (intraventricular) administration of BAAM (0.72 mg/g brain weight) resulted in no change in mean blood pressure or heart rate during a 20 min postinjection period. Biochemical analysis of heart tissue following central injections of BAAM showed little or no change in the number of BAR. There was, however, a significant decrease in the number of BAR in brain tissue.     

Immunosuppression by 2′-deoxycoformycin: Studies on the mode of administration

It was previously reported that continuous infusion of 2′-deoxycoformycin (DCF), a potent inhibitor of the enzyme adenosine deaminase (ADase), produces a severe immunodeficiency of both T and B lymphocytes (A. Tedde, M. E. Balis, S. Ikehara, R. Pahwa, R. A. Good, and P. P. Trotta, Proc. Nat Acad. Sci. USA 77, 4899 (1980)). In the present study the immunological and biochemical effects in C57BL/6J mice of daily single intraperitoneal injections of DCF are examined. Five days of daily injections of DCF at 0.4 mg/kg body wt/day produced both immunosuppression and lymphocytotoxicity. Parallel experiments indicated that continuous infusion of DCF at the lower dose of 0.3 mg/kg body wt/day resulted in a more potent depression of immune function and a greater lymphocytotoxicity. In particular we have observed after single injections: (i) A decrease in body weight of 4.7%, compared to 16.2% after continuous infusion; (ii) a decrease in differential lymphocyte count from 90.3% (normal) to 65.6%, compared to 31.4% after continuous infusion, and a decrease in leukocyte count (per mm²) from 5200 (normal) to 3800, compared to 2300 after continuous infusion, while no difference in other blood parameters, including erythrocyte number, hematocrit, and hemoglobin concentration, was observed; (iii) on histological examination, a depletion of T and, to a lesser extent, B lymphocytes, and an atrophy of lymph nodes, spleen, and thymus; and (iv) significant depression in blastogenesis induced by the T-cell mitogens concanavalin A and phytohemagglutinin and, to a lesser extent, by the B-cell mitogen Escherichia coli lipopolysaccharide. ADase was inhibited to a high degree ranging from 84 to 94% in lymphoid organs and erythrocytes from animals receiving DCF by either mode of administration. These data provide further direct support for the importance of ADase to proper lymphocyte function, and suggest that continuous infusion may be the preferred mode of administration in clinical trials of DCF as an antileukemic agent.