Design and synthesis of prodrugs of the rat selective toxicant norbormide

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, one major drawback of NRB as a viable rodenticide relates to an evolutionary aversion developed by the rat leading to sub-lethal dosing due to either its unpleasant 'taste' or rapid onset of effects. A series of NRB prodrugs were prepared in an effort to 'mask' this acute response. Their synthesis and biological evaluation (in vitro vasoconstrictory activity, in vitro hydrolytic and enzymatic stability and lethality/palatability in vivo) is described. Compound 19 displayed the most promising profile with respect to a delay in the onset of symptoms and was subsequently demonstrated to be significantly more palatable to rats.     

Prodrugs of N-dicarboximide derivatives of the rat selective toxicant norbormide

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, one major drawback of NRB as a viable rodenticide relates to an evolutionary aversion developed by the rat leading to sub-lethal dosing due to either its unpleasant ‘taste’ or rapid onset of effects. A series of NRB-derived prodrugs were prepared in an effort to ‘mask’ this acute response. Their synthesis and biological evaluation (in vitro vasoconstrictory activity, in vitro hydrolytic and enzymatic stability and lethality/palatability in vivo) is described. Prodrug 2 displayed the most promising profile with respect to a delay in the onset of symptoms and was subsequently demonstrated to be significantly more palatable to rats. Moreover, prodrug 25 was found to be largely accepted by rats in a choice trial, resulting in high mortality.     

Bioassay-guided isolation of a vasorelaxant active compound from Kaempferia galanga L.

Bioassay-guided fractionation was performed on a crude dichloromethane extract of Kaempferia galanga L. using chromatography techniques. Screening of the extract for biological activity started with the brine shrimp lethality bioassay, followed by the study of its antihypertensive activity on anaesthetized rats, which involved monitoring of the extract's effect on mean arterial blood pressure. The components of the fractions obtained from the separation procedures were analyzed using gas chromatography (GC). The yield of the CH(2)Cl(2) extract was 0.29% of the crude plant extract. Analysis of the data for brine shrimp lethality test using the Finney computer program showed that this extract exhibited potent bioactivity with an ED(50) value of 7.92+/-0.13 microgml(-1). Intravenous administration of the extract induced a dose-related reduction of basal mean arterial pressure (MAP) (130+/-5 mmHg) in the anaesthetized rat, with maximal effects seen after 5-10 min of injection. The gas chromatogram showed that the common compound in the active fractions obtained from the bioassay-guided fractionation of the CH(2)Cl(2) extract was ethyl cinnamate. This vasorelaxant active compound, ethyl cinnamate, was isolated as a colorless oil. Ethyl p-methoxycinnamic acid was also isolated as white needles but did not exhibit any relaxant effect on the precontracted thoracic rat aorta.     

Damage of rat liver microsomal mixed function oxidase system by carbon tetrachloride. In vivo study with selective inhibitor of lipid peroxidation.

Pretreatment of rats with 4-[4-N-sodium-N-(5-ethyl-1-thia-3,4-diazol-2-yl)sulfophenylamin o]-5- methoxy-1,2-benzoquinone (Q) before carbon tetrachloride intoxication inhibited lipid peroxidation by 85% but did not prevent cytochrome P-450 destruction, decrease of hydroxylase activity, and loss of the capability to bioactivate carbon tetrachloride in rat liver microsomes. Also no influence of Q on 10-day lethality was found. We conclude that covalent binding of free radical products of metabolic cleavage to various cellular structures is apparently the main damage factor of carbon tetrachloride hepatotoxicity rather than lipid peroxidation.     

Lethal effects of cocaine are reduced by the dopamine-1 receptor antagonist SCH 23390 but not by haloperidol

The prevalence of cocaine abuse has been associated with a host of medical complications and deaths. We investigated the effects of two dopamine antagonists with different affinities for dopamine-1 and dopamine-2 receptor subtypes on cocaine-induced lethality. Male Fischer-344 rats were given cocaine HCl (i.p.) and observed for lethality at 24 hrs. Cocaine was not lethal at 50 mg/kg and produced a steep dose-effect function from 60 to 100 mg/kg. Lethality was 88.9% at 100 mg/kg and the LD 50 was 79.7 mg/kg (95% CL: 74.8-84.9). Doses as high as 180 mg/kg failed to kill all rats. Lethality was often but not invariably associated with convulsions. Haloperidol (0.3-3 mg/kg i.p.) given 30 min prior to cocaine did not alter the lethal effects of cocaine but did reduce the lethality of methamphetamine. SCH 23390 (0.1-1 mg/kg i.p., 30 min prior) shifted the cocaine dose-effect function to the right at 0.3 mg/kg. Maximum protection was conferred by 0.3 mg/kg SCH 23390 where the LD 50 was increased to 100.1 mg/kg (95% CL: 91.5-109.5). Comparable protection was not observed if SCH 23390 was given 5 min after cocaine. These results suggest that dopamine receptors may play a role in the lethal effects of cocaine and that the D1 dopamine receptor subtype appears to be more relevant to lethality than the D2 subtype.     

Explaining social learning of food preferences without aversions: an evolutionary simulation model of Norway rats

Norway rats (Rattus norvegicus) transmit preferences for novel foods socially by smelling each other's breath. However, rats fail to learn aversions, acquiring a preference even if the rat whose breath they smell has been poisoned. Rats can distinguish between sick and healthy conspecifics and social learning of both preferences and aversions is present in other species - hence it is unclear why rats cannot learn aversions socially. We constructed an evolutionary simulation in which a population of rats foraged from a central location, exploiting food sites that could contain edible or toxic foodstuffs. We examined the relationship between toxin lethality and selection for individual versus social learning and discrimination between sick and healthy conspecifics in order to allow learning of both preferences and aversions. At low lethality levels individual learning was selected for and at intermediate levels we found social learning of both preferences and aversions. Finally, given high lethality levels the simulated rats would employ social learning but failed to learn aversions, matching the behaviour of real rats. We argue that Norway rats do not learn aversions socially because their environment may contain only highly lethal toxins which make interaction with a sick conspecific an extremely rare event.     

Interactions of drugs and radiation in haemopoietic tissue assessed by lethality of mice after whole-body irradiation

Drug-radiation interactions in haemopoietic tissue were assessed as the lethality of mice within 7-28 days after whole-body irradiation. The investigated drugs were adriamycin (ADM), bleomycin (BLM), cyclophosphamide (CTX), 5-fluorouracil (5-FU), methotrexate (MTX), mitomycin C (MM-C) and cis-diamminedichloroplatinum II (cis-DDP). The drugs were administered as single doses 15 min before graded doses of whole-body irradiation or at different intervals from 7 days before to 7 days after fixed radiation doses. ADM, CTX, 5-FU, MM-C and cis-DDP enhanced the radiation response when administered 15 min before irradiation. The dose effect factor (DEF) was 9.11 for 5-FU and in the range 1.25-1.59 for the other drugs. MTX administration 15 min before irradiation had no effect (DEF 1.00). However, MTX increased lethality if given 1-3 days after irradiation (DEF 1.21-1.76) and protected against lethality if given 1-3 days before irradiation (DEF 0.83). A similar time dependence was observed for ADM, CTX, 5-FU, MM-C and cis-DDP. Protection against lethality was not observed but in all these cases the lethality was significantly lower at administration 1-3 days before than 1-3 days after irradiation. A proper investigation of the effect of BLM was not possible as the combination of this drug and whole-body irradiation caused a high rate of gastrointestinal deaths.     

Japanese encephalitis virus (JEV): potentiation of lethality in mice by microwave radiation.

The expression of Japanese Encephalitis Virus (JEV) lethality in mice requires entry of the virus into the central nervous system. This entry is presumably through the capillary endothelial cells (CEC), because entry between CECs is inhibited by bands of circumferential tight-junctions. A viremic stage occurs during the first 4 to 5 days after JEV administration in mice, and both microwave radiation (2.45-GHz, continuous wave, 10-min exposure) and hypercarbia were employed to increase CEC permeability to JEV. Exposure to microwaves at power densities of 10-50 mW/cm2 resulted in a dose-dependent increase in JEV-induced lethality. Mice did not become tolerant or sensitized to microwave potentiation of JEV-induced mortality because 4 daily exposures at 10 or 50 mW/cm2 (SARS, approximately 24-98 W/kg) did not alter the lethality pattern to subsequent microwave radiation of JEV-exposed animals. Similarly, hypercarbia (5, 10, and 20% CO2) was observed to produce a dose-dependent increase in JEV-induced lethality. Both microwave radiation and hypercarbia are thought to promote pinocytosis in CNS capillary endothelial cells. This may be one mechanism by which they enhance JEV-induced lethality in adult Swiss-Cox mice.     

N-terminal hamartin-binding and C-terminal GAP domain of tuberin can separate in vivo

The Eker rat is an animal model of renal carcinogenesis and carries a transposon insertion in the Tsc2 (tuberous sclerosis-2) gene. We previously generated transgenic Eker rats and identified coding sequences in the Tsc2 gene that are responsible for suppression of renal carcinogenesis in Eker rats. Tsc2-RGH, a transgene that expresses the carboxy terminal region (amino acids 1425-1755) of the Tsc2 product (tuberin), partially suppressed renal carcinogenesis. However, Tsc2-DRG, which expresses a mutant tuberin lacking the carboxy-terminal region (Delta aa 1425-1755), did not suppress renal carcinogenesis. Here, we found that introduction of both Tsc2-RGH and Tsc2-DRG in Eker rats completely suppressed renal carcinogenesis and rescued homozygous (Tsc2(Ek/Ek)) mutants from embryonic lethality in a complementary manner. Co-introduction of Tsc2-RGH and Tsc2-DRG, but not introduction of either alone, efficiently suppressed phosphorylation of p70 S6K. Thus, the functional domains of N-terminal hamartin binding and C-terminal tumor suppression in tuberin can separate in vivo.     

Novel cyclohexene derivatives as anti-sepsis agents: synthetic studies and inhibition of NO and cytokine production

In order to develop an anti-sepsis agent, a series of cyclohexene derivatives were synthesized and evaluated for their biological activities. Through modification of the sulfonamide spacer moiety depicted by formula II, it was found that the benzylsulfone derivative 10a had potent inhibitory activity against the production of NO. Further modifications of the phenyl ring, ester moiety, and benzyl position of benzylsulfone derivatives III were carried out. Among these compounds, (R)-(+)-10a and (6R, 1S)-(+)-22a showed strong inhibitory activity not only against NO production but also against inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in vitro. Furthermore, (R)-(+)-10a and (6R, 1S)-(+)-22a protected mice from LPS-induced lethality in a dose-dependent manner.     

Transposon insertion mutagenesis of a genetic region encoding serum resistance in an 80 kb plasmid of Salmonella dublin

Using transposon insertion mutagenesis with Tn1 or Tn5, we obtained Salmonella dublin mutant strains that showed either diminished serum resistance (five mutants) or diminished mouse lethality (two mutants). Detailed restriction cleavage analysis to determine the single sites of transposon insertion in an 80 kb plasmid (pTE800) indicated that a region for serum resistance was located within a 3.0 kb region of the SalI cleavage fragment 5 and the HindIII fragment 2, while the region for mouse lethality was within a 6.0 kb region of the SalI fragment 2 and the HindIII fragment 1. When the Tn1-containing SalI fragment 5 was reconverted, by homologous recombination, to the original SalI fragment 5 (9.6 kb), serum resistance was recovered to the same level as that of a parent strain 52401. Moreover, the change in the serum resistance correlated with changes in the neutral sugar composition of the LPS. The mutation in the plasmid in strain TE4-55 that gave diminished mouse lethality was also reversed by recombination with the cloned SalI fragment 2 (15.0 kb), with concomitant recovery of mouse lethality. These results indicate that the genetic region for serum resistance is different from that for mouse lethality, and that the gene for serum resistance is closely involved with the expression of the neutral sugar composition of the LPS of S. dublin.     

Acute heat stress protects rats against endotoxin shock.

The purpose of this study was to determine 1) whether prior (24-h) heat stress could render rats cross-resistant to the lethal activity of bacterial lipopolysaccharide (LPS) and 2) whether this acquired state of resistance is associated with endotoxemia during the heat stress event. Four groups (n = 7/group) of rats were examined: 1) saline treated, 2) LPS treated, 3) heat stressed and saline treated, and 4) heat stressed and LPS treated. Saline or LPS (Escherichia coli, serotype 0111:B4, 20 mg/kg body wt) was given intravenously 24 h after exposure to heat (ambient temperature 47-50 degrees C, relative humidity 30%) for heat-stressed rats and at the same time of day for nonheated rats; survival was monitored for 48 h. Thermal responses were similar (P > 0.05); values for maximum core temperature (Tc) and time above Tc of 40 degrees C were 42.7 +/- 0.1 and 42.6 +/- 0.1 degrees C (SE) and 44.0 +/- 2.1 and 47.9 +/- 3.7 (SE) min for the heat-stressed saline-treated and heat-stressed LPS-treated rats, respectively. Administration of LPS to nonheated rats resulted in 71.4% (5 of 7 rats) lethality. In contrast, all (7 of 7) rats subjected to a single nonlethal heat stress event 24 h before LPS treatment survived (P < 0.05). Endotoxin was not detected in arterial plasma immediately after heat stress in rats (n = 6) exposed to a Tc of 42.9 +/- 0.1 degrees C. These findings demonstrate that acute heat stress can protect rats from the lethal activity of LPS.     

Endothelin-induced sudden death and the possible involvement of platelet activating factor (PAF)

Endothelin (5 nmol/kg, i.v.) caused a transient hypotension followed by a lasting hypertension in rats. However, an abrupt fall in the blood pressure was observed in most rats 6 to 30 min after the injection of endothelin and sudden death followed with lethality noted over 60 min. An abnormal electrocardiogram (ECG) (ventricular arrhythmias) was observed in rats injected with endothelin. Endothelin (i.v.) also caused sudden death in mice. Pretreatment (5 or 60 min) with specific PAF antagonists, CV-6209 (0.1-3 mg/kg, i.v.) and WEB 2086 (30 mg/kg, p.o.), and a calcium channel blocker, diltiazem (60 mg/kg, p.o.) prevented death and attenuated the ECG changes induced by endothelin, but CV-6209 did not prevent the blood pressure changes induced by endothelin. CV-6209 (0.5-3 mg/kg, i.v.), WEB 2086, diltiazem and dexamethasone (5 mg/kg, i.v.) protected mice against the death induced by endothelin. On the other hand, aspirin (cyclooxygenase inhibitor, 100 mg/kg, p.o.) did not protect mice from the death. Thus, endothelin is a highly toxic peptide with cardiotoxic effects, and PAF may be involved in the pathogenesis of the sudden death.     

Restructured transactivation domain in hamster AH receptor

Hamsters and Han/Wistar (Kuopio; H/W) rats show peculiarly selective responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). They are extremely resistant to its acute lethality but sensitive to, e.g. , enzyme induction. The biological effects of TCDD are mediated by the AH receptor (AHR). Recent studies on H/W rat AHR discovered a remodelled transactivation domain which appears to be critical for the TCDD resistance of these animals. Here, molecular cloning and sequencing of hamster AHR reveals another type of restructured transactivation domain. In hamsters, the functionally pivotal Q-rich region is substantially expanded and enriched in glutamine compared with all other AHRs cloned to date. By contrast, the amino-terminal end is highly conserved, which is in agreement with the H/W rat AHR. Because of the additional material in the transactivation domain, hamster AHR protein is larger than that in rats or mice, but the pattern of AHR mRNA expression in tissues is similar.     

Production of monoclonal antibody to Clostridium difficile toxin A which neutralises enterotoxicity but not haemagglutination activity

Nine monoclonal antibodies (mAb) to Clostridium difficile toxin A were produced. The isotype of one mAb (37B5) was IgG2b, kappa, and that of the other eight mAbs was IgM, kappa. Immunoblot analysis after non-denatured PAGE showed that with the exception of one mAb (112G6) all mAbs gave a positive reaction with the 540 kDa band of toxin A. Immunoblot analysis showed that four mAbs (2E15, 3B4, 37B5 and 49C4) gave a positive reaction with the 240 kDa major band of toxin A. In neutralisation tests with these mAbs for enterotoxicity, mouse lethality, haemagglutination activity and cytotoxicity, 37B5 neutralised enterotoxicity in a rabbit ileal loop response test but did not neutralise any other biological activities. None of the other eight mAbs showed any neutralising activities at all.     

Potassium nitrate effect on the development of neurological disorders in experimental brain ischemia

Effect of potassium nitrate (KNO₃) on the dynamics of neurological disorders and lethality in rats as sequelae of brain ischemia induced by a single-step bilateral common carotid artery occlusion were investigated in Wistar rats. KNO₃ at a dose of 5 mg/1000 g administered 60 min prior to the occlusion of the two carotid arteries reliably reduced the severity of neurological disorders and lethality in rats.     

Identification of novel splice variants of ARNT and ARNT2 in the rat

Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long-Evans (Turku/AB) (L-E); the LD50 values for these two strains are >9600 and 10-20 microg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L-E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3(') end of exon 6 or 5(') end of exon 11, or with an insertion at 5(') end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5(') end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5(') end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.     

Ventilatory long-term facilitation is greater in 1- vs. 2-mo-old awake rats.

Respiratory long-term facilitation (LTF) declines in middle-aged vs. adult male rats. Chronic intermittent hypoxia (CIH; 5 min 11-12% O2/5 min air, 12 h/night, 7 nights) enhances LTF in adult rats. However, LTF in immature rats and the effect of early CIH are unevaluated. The present study compared LTF in 1- and 2-mo-old rats and examined the effect of neonatal CIH (initiated at 2 days after birth) on the LTF. Ventilatory LTF, elicited by 5 (protocol 1) or 10 (protocol 2) episodes of poikilocapnic hypoxia (5 min 12% O2/5 min air), was measured twice by plethysmography on the same male conscious rat when it was 1 and 2 mo old. In untreated (without CIH) rats, both resting ventilation (54.7 +/- 0.6 vs. 43.0 +/- 0.2 ml.100 g(-1).min(-1)) and hypoxic ventilatory response (131 +/- 4 vs. 66 +/- 3% above baseline) were greater in 1- vs. 2-mo-old rats. Protocol 1 elicited LTF in 1-mo-old (12.5 +/- 1.0% above baseline) but not 2-mo-old rats. Protocol 2 elicited a greater LTF in 1-mo-old (24.3 +/- 0.8%) vs. 2-mo-old rats (18.2 +/- 0.5%). In CIH-treated rats, protocol 1 also elicited LTF in 1-mo-old (13.1 +/- 1.5%) but not 2-mo-old rats. Protocol 2 elicited LTF in both age groups, but LTF was enhanced by the CIH only in 1-mo-old rats (28.8 +/- 0.9%). These results suggest that ventilatory LTF and hypoxic ventilatory response are greater in male rats shortly before their sexual maturity and that the neonatal CIH somewhat enhances ventilatory LTF approximately 3 wk after CIH, but this enhancement does not last to adulthood.