Biochemical, histological, and ultrastructural changes in rat and mouse liver following the administration of trichloroethylene: possible relevance to species differences in hepatocarcinogenicity

Trichloroethylene (TRI), administered by gavage for 10 consecutive days, at doses of 500 to 1500 mg/kg body wt increased liver weight (175% of control), decreased hepatic DNA concentration (66% of control), and increased the synthesis of DNA (500% of control; as measured by [3H]dT incorporation) in B6C3F1 mice and Alderley Park mice. Similar treatment of Osborne-Mendel rats or Alderley Park rats resulted in smaller increases in liver weight (130% of control) and decreases in DNA concentration (83% of control). No effect of TRI on DNA synthesis was seen in rats. The increased DNA synthesis in the mouse was not apparently due to regenerative hyperplasia since no signs of necrosis were seen. Furthermore the increased [3H]dT incorporation probably represented semiconservative replication of DNA and not repair, since a parallel increase of mitotic figures was observed. Hence, the liver growth noted after TRI administration appears to be due to liver cell enlargement (hypertrophy) in the rat, but both hypertrophy and hyperplasia (cell proliferation) in the mouse. An important observation has been that TRI induced the peroxisomal enzyme activities, catalase, and cyanide-insensitive palmitoyl-CoA oxidation (147 and 786% of control, respectively), in mice but not in rats. Furthermore, increases in peroxisome volume density (up to 1110% of control) were observed in mice receiving TRI. These observations lead us to suggest that the species difference in hepatocarcinogenicity of TRI, seen between the rat and mouse, is possibly due to a species difference in peroxisome proliferation and cell proliferation, the peroxisome proliferation leading to increased reactive oxygen species and DNA damage, and the cell proliferation then acting to promote this lesion.     

Lack of induction of rat liver mixed-function oxidases after chronic administration of high brotizolam doses to rats

Rats were treated with 10, 200 or 400 mg/kg brotizolam (Lendormin) for 4 weeks, then liver microsomes were prepared and the in vitro transformation of several model substances studied. Furthermore, after similar treatment of rats, the metabolite pattern in the plasma was studied using [14C]brotizolam as a marker. Finally the same investigations were performed after pretreating the rats with the enzyme inducers, phenobarbital or 3-methylchol-anthrene, for 3 days instead of brotizolam. The amount of microsomal protein in the rat liver was increased after all 3 doses of brotizolam, the liver weight after the highest dose only. Activity of the flavoenzyme NADPH cytochrome-c reductase was the only enzyme activity increased after 200 and 400 mg/kg brotizolam, whereas cytochrome P-450 content decreased after 400 mg/kg brotizolam. Activities of the mixed-function oxidases studied were not changed at all. Marked changes after brotizolam administration were seen in the metabolite pattern. The higher doses led to reduced amounts of both of the very polar metabolites. Simultaneously metabolite We 964 (= brotizolam hydroxylated at the methyl group) and the unchanged brotizolam increased several-fold. Treatment of rats with phenobarbital or 3-methylcholanthrene showed the typical but different changes in enzyme activities. The metabolite pattern of brotizolam, however, was not changed. From the results it is concluded that a 4-week treatment of rats with up to 400 mg/kg brotizolam causes no induction of mixed-function oxidases in the liver. The changes of the metabolite pattern described can be discussed as an effect of liver enzyme saturation.     

Tolerance following the repeated administration of high doses of phencyclidine: No relation to central catecholamine depletion

The tolerance which develops following the repeated administration of phencyclidine has been attributed to both pharmacological and behavioral adaptations. In the present study, the possibility that neurochemical alterations induced by the repeated administration of phencyclidine might account for some portion of the observed tolerance was examined. Specifically, it was postulated that the repeated administration of high doses of phencyclidine might result in long lasting depletion of central catecholamine stores and that this neurochemical perturbation might result in tolerance to the drug's effects. It was observed that phencyclidine disrupted the performance of rats on a fixed-interval schedule maintained by water presentation. The repeated administration of high doses of phencyclidine (but not of a comparable volume of saline) during a period when subjects were not engaged in the fixed-interval task resulted in the development of a long lasting tolerance, but this tolerance could not be accounted for in terms of a depletion of central catecholamine stores.     

Clinical pharmacology of moclobemide during chronic administration of high doses to healthy subjects

The objectives of this study were to assess the tolerability, safety, pharmacodynamics and pharmacokinetics of high-dose moclobemide in healthy subjects. Two sequential groups of six male and six female subjects (eight on active treatment, four on placebo) received for 8 days moclobemide 450 mg b.i.d. and 600 mg b.i.d., respectively. Intravenous tyramine pressor tests were conducted at baseline, at the beginning of treatment and at steady state. Oral tyramine pressor tests with 50, 100 and 150 mg tyramine were conducted under steady-state conditions. Pharmacokinetic parameters of moclobemide and two of its metabolites in plasma and urine were determined after the first and last dose of moclobemide. The incidence and intensity of adverse events was dose-dependent. The most frequently reported adverse events were insomnia, headache, dizziness and dry mouth. The IV tyramine pressor sensitivity during both moclobemide dosing regimens was enhanced 3 to 4-fold. Intake of tyramine 50 mg did not result in systolic blood pressure increases greater than 30 mmHg. With regard to blood pressure increases, tyramine 100 mg is still compatible with moclobemide 450 mg b.i.d. but not with 600 mg b.i.d. The clearance of moclobemide decreased by about 60% on multiple dosing, but no differences were found between both dosing regimens. The urinary excretion of the N-oxide metabolite doubled during multiple dosing. In conclusion, the maximum tolerated dose of moclobemide in healthy subjects is 600 mg b.i.d. provided the tyramine content in a meal is not higher than 50 mg. Received: 27 October 1997/Final version: 31 March 1998     

Perchloroethylene-induced rat kidney tumors: an investigation of the mechanisms involved and their relevance to humans

Lifetime exposure to perchloroethylene by inhalation has been shown to cause a low incidence of renal tumors in male rats. The mechanisms responsible for the induction of these tumors have been investigated following exposure of rats to perchloroethylene by oral gavage (1500 mg/kg for up to 42 days) or by inhalation (400 ppm for 28 days). Comparisons have been made between rats and mice in vivo and between rats, mice, and humans in vitro. High doses of perchloroethylene given by gavage have been shown to be toxic to the rat kidney, causing increases in urinary markers of kidney damage. A marked accumulation of protein droplets (alpha-2u-globulin) was seen in the P2 segment of the kidney proximal tubules. This response were not seen after inhalation exposure to 400 ppm perchloroethylene for 28 days and hence may not be associated with the tumors seen at this dose level. Protein droplet formation was seen after exposure to 1000 ppm perchloroethylene, suggesting that 400 ppm is below the threshold dose required to induce this response. Perchloroethylene has been shown to be metabolized by glutathione conjugation in the liver, resulting in the formation of a mutagenic cysteine conjugate which is activated by the kidney enzyme beta-lyase. Levels of the mercapturic acid of perchloroethylene have been compared in rat and mouse urine. The enzyme kinetics of hepatic glutathione conjugation and renal beta-lyase activation have been compared in rat, mouse, and human tissues in vitro. Results of these studies are consistent with the rat being the species susceptible to kidney tumors. Although human kidney was shown to contain beta-lyase, glutathione conjugation of perchloroethylene could not be detected in human liver. Perchloroethylene-induced male rat kidney tumors may be a result of chronic toxicity, protein droplet nephropathy, and genotoxicity from the beta-lyase pathway. These mechanisms appear to have little relevance to humans.     

Astrocytes: adaptive responses to low doses of neurotoxins

This article assesses how astrocytes respond to numerous endogenous agonists and a wide variety of chemical stressors, including well-known neurotoxic agents such as lead and methylmercury, and drugs, within the context of dose-response relationships. In general, astrocytes displayed biphasic dose-response relationships from exposure to neurotoxic agents with temporal and quantitative features similar to hormetic dose responses. While the low-dose stimulatory responses have been viewed as a manifestation of a toxic response, this perspective is being broadly reconsidered and in some cases reinterpreted as being an indicator of an adaptive/protective response. These dose-response patterns are likely to have significant implications in a wide range of conditions affecting normal developmental processes, tumor development, adaptive responses to numerous environmental neurotoxins, and strategies for drug development for the treatment of neurodegenerative conditions.     

Cardiovascular responses to intrathecal administration of L- and D-baclofen in the rat.

D- and L-baclofen were given intrathecally at the T2 spinal level in the anaesthetized rat. D-Baclofen, in doses of 7, 35 and 70 nmol produced graded increases in arterial pressure but heart rate remained unaffected. Responses appeared within 30 s, peaked at 2 min and decayed over the next 5 min. Injection i.v. of 70 nmol of D-baclofen failed to alter arterial pressure or heart rate. In contrast, intrathecal administration of L-baclofen decreased both arterial pressure and heart rate. The amplitude and time course of the effects depended on the dose used; 700 nmol of L-baclofen had stronger and longer effects than those induced by 70 nmol, while 7 nmol had no effect. (I.v. injection of 70 nmol of L-baclofen had similar effects to intrathecal administration but with different time course and amplitude.) When given at the T9 level at doses of 70 nmol, D- and L-baclofen had effects similar to those observed at the second thoracic level. Effects of intrathecal administration of D- and L-baclofen at T2 were prevented by pretreatment with either hexamethonium (10 mg/kg i.v.) or lidocaine (25 microliters of a 1% solution, intrathecally). The results suggest that D- and L-baclofen-sensitive receptors in the spinal cord are involved in regulating sympathetic output in pathways to the vessels and/or to the heart. In addition, our results suggest that D- or L-baclofen may not act via classical GABAB receptors or that two types of GABAB receptor exist in spinal sympathetic pathways.     

Absence of central nervous system and hypothermic effects after single oral administration of high doses of oseltamivir in the rat

Oseltamivir is widely used for the treatment and prophylaxis of influenza. Renewed interest in the central nervous system (CNS) tolerability profile of oseltamivir has been triggered by the reports of neuropsychiatric adverse events in patients with influenza. In addition, a recent pre-clinical study in rodents suggested a hypothermic effect of oseltamivir. The current studies investigated the CNS effects, body temperature effect and toxicokinetic profile of oseltamivir in rats. The CNS/temperature study included three groups receiving oseltamivir (500, 763 and 1000 mg/kg free base by oral gavage), one vehicle/control group and one reference group (D-amphetamine, 10 mg/kg). CNS parameters (behaviour, motor activity and co-ordination and sensory/motor reflex responses) and rectal temperature were measured at baseline and at five intervals until 8 hr after dosing. In the toxicokinetic study, rats received oseltamivir by oral gavage at 763 or 1000 mg/kg free base. Plasma, cerebrospinal fluid (CSF) and perfused brain concentrations of oseltamivir and its active metabolite, oseltamivir carboxylate (OC), were measured until 8 hr after dosing. Median scores for CNS parameters were similar in controls and animals receiving oseltamivir at all time points. Oseltamivir had no physiologically relevant effect on body temperature, but induced a short-lived and small dose-independent decrease in temperature in all active treatment groups at 1 hr after dosing only. Plasma concentrations of OC were higher than of oseltamivir, but the reverse was true in CSF and brain. CNS penetration was low for both moieties. In rats, oseltamivir at supratherapeutic doses up to 1000 mg/kg free base did not exert any effects on CNS function or hypothermic effects and led to limited CNS exposure, resulting in large safety margins.     

The enhanced biliary secretion of a taurine conjugate in the rat after intraduodenal administration of high doses of fenclozic acid

1. The metabolic fate of [¹⁴C]fenclozic acid (ICI 54450) in rat was determined after intraduodenal administration at different doses.

2. Increasing the dose from 2 to 100mg/kg resulted in a five-fold increase in drug-related material secreted in bile.

3. At a dose of 2?mg/kg the taurine conjugate was a relatively minor metabolite, whereas at 100mg/kg this conjugate was the major metabolite in bile and urine.

4. Enhanced biliary secretion of the taurine conjugate in rats receiving multiple doses of fenclozic acid results in exposure of the intestinal cells to much greater concn. of drug-related metabolites.     

Beta-adrenoceptor responses to high doses of inhaled salbutamol in patients with bronchial asthma.

1. Fourteen asthmatics (mean +/- s.e. mean baseline FEV1 62 +/- 6% of predicted) were given cumulative doubling doses of salbutamol by metered-dose inhaler as follows: 100 micrograms, 200 micrograms, 500 micrograms, 1000 micrograms, 2000 micrograms, 4000 micrograms. 2. Airways, tremor, haemodynamic and cyclic AMP responses were measured at each dose increment (made every 20 min). 3. There was a linear log dose-response relationship for each airways parameter (FEV1, VC, sGaw, FEF 50%). The plateau in the dose-response curve was not reached within our dose range. These changes were also mirrored in cyclic AMP responses. 4. There was a wide range in maximum airways response expressed in terms of absolute increase over baseline (95% confidence intervals: delta FEV1 667-1483 ml; delta VC 689-1695 ml; delta sGaw 0.92-4.50 s-1 kPa-1; delta FEF 50% 0.94-2.15 l s-1). Patients with a lower baseline showed a greater response in terms of percent increase in FEV1 (r = -0.83, P less than 0.001). There was however, no correlation between baseline airway calibre and the dose required for maximum bronchodilatation. 5. There were objective increases (mean +/- s.e. mean) in both heart rate (maximum delta HR of 14 +/- 5 beats min-1 at 4000 micrograms) and tremor power (maximum delta Tr of 115 +/- 44% at 2000 micrograms). These were not dose limiting side-effects as subjective symptoms were infrequent at higher doses. 6. Higher than conventional doses of salbutamol given by metered-dose inhaler may produce a distinct improvement in airways response without significant side-effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epoetins: differences and their relevance to immunogenicity

Recombinant human erythropoietin (epoetin) is a highly active molecule and as such is used at very low therapeutic concentrations that require stabilisation. Commercially available epoetins differ in the stabilisers used in their formulations, which result in variations between epoetin preparations in storage and handling requirements. The stability and solubility of the epoetins are also affected by differences in the carbohydrate moieties that exist between them. However, it is the difference in stabilising agents that is thought to be the major cause of the upsurge in pure red cell aplasia (PRCA) cases observed predominantly with one epoetin alfa formulation, Eprex (Johnson & Johnson). In 1998 the European formulation of Eprex was changed with the replacement of human serum albumin (HSA), by polysorbate 80 and glycine. This formulation change coincided with an increased incidence of PRCA. In contrast, the incidence of PRCA has remained low with other HSA-containing epoetin alfa products and with epoetin beta. Therefore, it appears that the change in Eprex formulation has resulted in reduced protein stability and increased immunogenicity.     

Epoetins: differences and their relevance to immunogenicity

SUMMARY

Recombinant human erythropoietin (epoetin) is a highly active molecule and as such is used at very low therapeutic concentrations that require stabilisation. Commercially available epoetins differ in the stabilisers used in their formulations, which result in variations between epoetin preparations in storage and handling requirements. The stability and solubility of the epoetins are also affected by differences in the carbohydrate moieties that exist between them. However, it is the difference in stabilising agents that is thought to be the major cause of the upsurge in pure red cell aplasia (PRCA) cases observed predominantly with one epoetin alfa formulation, Eprex (Johnson & Johnson). In 1998 the European formulation of Eprex was changed with the replacement of human serum albumin (HSA), by polysorbate 80 and glycine. This formulation change coincided with an increased incidence of PRCA. In contrast, the incidence of PRCA has remained low with other HSA-containing epoetin alfa products and with epoetin beta. Therefore, it appears that the change in Eprex formulation has resulted in reduced protein stability and increased immunogenicity.     

Facilitation of self-stimulation with high doses of amphetamine in the rat

Rats were trained to self-stimulate by interrupting a photobeam and brain stimulation was maintained for as long as the beam of light was broken. d-Amphetamine sulphate was then administered and response rate and total duration of stimulation were recorded. Both response rate and total duration were elevated by 1.0, 2.0, and 5.0 mg/kg dosages. The 1.4 s/response duration observed with saline was elevated to 2.0 s/response with 2.0 and 5.0 mg/kg doses. It was concluded that amphetamine's effects on self-stimulation are at least partially determined by the response requirements of the task employed.     

Cardiovascular responses to noradrenaline in the rat before and after administration of various anaesthetics

1. Cardiovascular responses to intravenously administered noradrenaline were monitored in the rat in the conscious state and again following anaesthesia. Injections and pressure recordings were made via permanently implanted venous and arterial catheters.2. The pressor response to noradrenaline was inhibited by diethyl ether, urethane and deep pentobarbitone anaesthesia. It was unchanged by ketamine and was potentiated by chloralose and light pentobarbitone anaesthesia.3. Reflex bradycardia was potentiated by chloralose, blocked by ketamine and reduced by pentobarbitone. It was either reduced or unchanged by ether or urethane.     

Maternal high fat diet during the perinatal period alters mesocorticolimbic dopamine in the adult rat offspring: reduction in the behavioral responses to repeated amphetamine administration

Rationale Early environment can shape the development and function of the mesocorticolimbic dopamine (DA) system and represents a possible risk factor for adult pathologies. One critical variable in the early environment is nutrition, and exposure to high fat (HF) in adulthood is known to change this DA system. Objectives We tested whether perinatal HF intake in rats could have long-term effects on DA function and behavior in adult offspring. Materials and methods Rat dams were fed either a control (5% fat, CD) or high fat (30% fat, HF) diet during the last week of gestation and lactation, and adult offspring were tested (PND 56–90) after weaning on CD. Locomotor responses to acute and repeated doses of d-amphetamine (AMP, 0.75 mg/kg bw) were determined as were indices of DA function in the ventral tegmental area (VTA), nucleus accumbens (NAc), and the prefrontal cortex (PFC). Results Adult HF offspring displayed increased tyrosine hydroxylase expression in the VTA and NAc and significant increases in DA and DOPAC content in the NAc, suggesting an elevated DA tone in this target field. In the NAc, there were no significant changes in D1, D2 receptors, or DA transporter (DAT) levels between diet groups. Perinatal HF feeding reduced AMP-induced locomotion and behavioral sensitization to AMP, suggesting that early diet might have caused long-lasting desensitization of postsynaptic receptor mechanisms in the NAc. Conclusions Our results demonstrate that both synthetic activity in VTA neurons and the responsiveness of accumbens DA neurons is altered by maternal nutrition. These effects subside long after termination of exposure to the HF diet.     

Differential neurotensin responses to low and high doses of methamphetamine in the terminal regions of striatal efferents

Neurotensin is a neuropeptide associated with basal ganglia dopaminergic neurons. Because levels of neurotensin in striatal tissue are differentially affected by low or high doses of methamphetamine, we employed microdialysis to assess the dose-dependent effects of methamphetamine on neurotensin release from the terminals of striatonigral and striatopallidal neurons. A low (0.5 mg/kg), but not high (10 mg/kg), dose of methamphetamine significantly increased nigral extracellular levels of neurotensin. The low-dose effect on extracellular nigral neurotensin levels was blocked by pretreatment with either a dopamine D1 or D2 receptor antagonist. In the globus pallidus, only half of the animals demonstrated increased neurotensin release after the low dose of methamphetamine. These findings suggest that low and high doses of methamphetamine differentially affect the release of neurotensin from the terminals of striatonigral neurons and that both dopamine D1 and D2 receptor activation contributes to the low-dose methamphetamine effects in the substantia nigra.     

Effects of the administration of two doses of sulpiride on the behaviour of the rat

The effect of intraperitoneal administration of 40 and 80 mg/kg of sulpiride on the behaviour of the rat was studied. At 40 mg/kg sulpiride acted as an anxiolytic agent since it slightl) increased the exploration of the internal areas of the open field, signilicantly decreased defecations in this lest. facilitated the approach to the goal box in a conflictivc situation and had no revelant effects on the acquisition and extinction of an active avoidance response. At the dose of 80 mg/kg, a decrease in the exploratory behaviour in the open field (without an increment in defecations), an impairment in the acquisition and an irregular performance of the active avoidance response in the shuttle box. and a decrease of inter-trial responses in the shuttle box occurred. A recovery in the active avoidance response was observed when longer delays for response were allowed. The effects at that dose are different from those of neuroleptics and may he interpreted as due to a general depression on behaviour.     

Protein binding and pharmacokinetics of lincomycin following intravenous administration of high doses

High-dose infusions of lincomycin 600, 1,200, and 2,400 mg were administered to 14 healthy, adult men. Using model-independent pharmacokinetics, it was found that the half-life, mean residence time, and steady-state volume of distribution of total drug increased with dose, whereas the same parameters remained unchanged for the unbound lincomycin. Although the mean clearance value for total drug increased, this change fell short of being significant at the 5% level and was associated with a decrease in unbound clearance following administration of the 2,400 mg dose. Protein binding studies using ultrafiltration gave direct evidence of saturable serum protein binding and indicated that binding involved at least two distinct classes of binding sites.