Hepatic metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat and guinea pig

Marked interspecies variability exists in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with the rat having an LD50 about 25-fold greater than the guinea pig. The metabolism of TCDD was examined by incubating hepatocytes isolated from these animals with purified [14C]TCDD (2.2 microM) for 8 hr. Over the 8-hr incubation, cytochrome P-450 content and ethoxyresorufin O-deethylase and benzphetamine N-demethylase activities were well maintained, indicating the functional viability of the hepatocytes. Quantitative differences were observed in the rate of [14C]TCDD metabolism, with hepatocytes from control rats metabolizing TCDD at a rate 2.8-fold greater than hepatocytes from control guinea pigs. The role of the hepatic cytochrome P-450-448-dependent monooxygenase system in the metabolism of TCDD was examined through the use of hepatocytes isolated from animals pretreated with either TCDD (5 micrograms/kg, ip; 72 hr prior to hepatocyte isolation) or phenobarbital (80 mg/kg, ip X 3 days; 24 hr prior to isolation). The rate of [14C]TCDD metabolite formation in hepatocytes from TCDD pretreated guinea pigs (0.26 +/- 0.14 pmol mg cell protein-1 hr-1) was unchanged from the control rate (0.25 +/- 0.07), while the rate in hepatocytes from TCDD pretreated rats (2.26 +/- 0.43 pmol mg-1 hr-1) was 3.2-fold greater than control (0.70 +/- 0.10) and nine times greater than in hepatocytes from TCDD-pretreated guinea pigs. In addition, significant differences were observed in the profiles of the metabolites formed by hepatocytes from TCDD-pretreated rats and guinea pigs. On the other hand, phenobarbital pretreatment produced little change in the rate of [14C]TCDD metabolism in rat hepatocytes (0.98 +/- 0.13 pmol mg-1 hr-1). These results suggest that TCDD may be metabolized by a TCDD inducible form of cytochrome P-448 which is expressed in the rat but not in the guinea pig. Furthermore, the differences in the hepatic metabolism of TCDD in the rat and guinea pig and in the ability of TCDD to induce its own rate of metabolism may play a major role in explaining the varying susceptibility of these species to the acute toxicity of TCDD.     

Solvent ototoxicity in the rat and guinea pig

There is clear evidence that aromatic solvents can disrupt the auditory system in humans and animals. As far as animal models are concerned, solvent-induced hearing loss seems to be species-dependent. Indeed, most published data have been obtained with the rat, which shows mid-frequency cochlear deficits, whereas the guinea pig does not show any permanent hearing loss after solvent exposure. In the current investigation, the effects of two solvents, toluene (600 ppm) and styrene (1000 ppm), were studied in both Long-Evans rats and pigmented guinea pigs exposed 6 h/day for 5 consecutive days. Cochlear function was tested by using distortion product otoacoustic emissions (DPOAE) measured prior to the solvent exposure, 20 min after the end of the exposure and successively at 2 and 4 weeks post-exposure. In addition to cochlear testing, solvent concentrations in blood and urinary metabolites were measured. A cochlear histological analysis was performed at the end of the experiment. No decrease in DPOAE amplitude was observed in the guinea pig, even immediately following the end of exposure. The rat model showed severe disruption of auditory function and cochlear pathology, whereas the guinea pig had no disruption of DPOAE or cochlear pathological alterations. Therefore, the vulnerability of the cochlear function was strictly dependent on the species. As expected, an important difference in the styrene concentration in blood was observed: the solvent concentrations were fourfold higher in the rat than in the guinea pig. Therefore, it is clear that a pharmacokinetic or an uptake difference might explain the difference in susceptibility observed between the two species. Moreover, the metabolism pathways of the solvents were different depending on the species. Attempts to explain differences of vulnerability between the rat and guinea pig are addressed in the present paper.     

Bromobenzene metabolism in the rat and guinea pig

The metabolism of bromobenzene was studied in the rat and guinea pig with respect to three considerations: the dose and species dependence of 3-bromophenol excretion; the formation of methylthio analogs of dihydrodiols and catechols; and the identification of acidic bivalent sulfur metabolites. In the guinea pig, 3-bromophenol was the major monohydric phenolic metabolite under conditions of both relatively low and relatively high dosage. In the rat, 3-bromophenol and 4-bromophenol were formed in approximately equal amounts. 2-Bromophenol was a minor metabolite in both species. Methylthio analogs of dihydrodiols were found as guinea pig, but not rat, metabolites. Two di(methylthio)dihydroxytetrahydrobromobenzene metabolites were excreted by the rat but not by the guinea pig. These methylthio compounds have not been reported in earlier studies of bromobenzene metabolism. In the guinea pig, the acidic urinary metabolites were a mercaptoacetate, a mercaptolactate, and a mercapturate. In the rat, the acidic metabolites were a mercapturic acid and premercapturic acids. This species difference in urinary acids indicates a difference in acetylation/deacetylation processes for cysteine conjugates.     

The role of toxicokinetics in xylene-induced ototoxicity in the rat and guinea pig

In the rat, some aromatic solvents cause a high level of ototoxicity that is characterized by damage to outer hair cells in the cochlea, which results in irreversible hearing loss. However, there is a vast difference in their potency. Among the three isomers of xylene, only para-xylene has been shown to be ototoxic in the rat. Moreover, all the species do not show the same susceptibility to ototoxic solvents, the rat being the most susceptible and the guinea pig seeming resistant to this ototoxic effect. The objective of the study was to determine whether toxicokinetic factors could explain the differences in ototoxicity observed among the three isomers of xylene in the rat and the species-dependent ototoxicity in the rat and the guinea pig. Blood and brain concentrations of each isomer were monitored in Sprague-Dawley rats treated orally by gastric intubation with a single dose or a 10 day-repeated treatment of 8.47 mmol/kg (an ototoxic dosage for para-xylene) of each isomer. Moreover, histology of the cochlea was carried out and the toxicokinetics of meta-xylene was monitored in rats treated with a single dose or a 10 day-repeated treatment of 16.94 mmol/kg meta-xylene, a non-ototoxic isomer. Similarly, histology of the cochlea was carried out and the toxicokinetics of para-xylene was followed in guinea pigs treated by gavage with a single dose or a 10 day-repeated treatment of 8.47 mmol/kg para-xylene. Finally, the blood and brain concentrations of para-xylene were measured in both the rats and the guinea pigs after a 4-h exposure to 1800 ppm of para-xylene. Among the three isomers studied, para-xylene yielded the highest blood and brain concentrations in the acutely and repeatedly exposed rats. When given a high dosage of meta-xylene (16.94 mmol/kg), the rats showed blood and brain concentrations of meta-xylene in the same order as those obtained with 8.47 mmol/kg para-xylene, but no outer hair cell loss was observed. No outer hair cell loss was observed in the guinea pigs treated with para-xylene. Whatever the exposure pattern, the blood and brain concentrations of para-xylene in the rats were 3.1-9.5 times higher than those measured in the guinea pigs. These results indicate that toxicokinetic factors cannot explain the differences in ototoxicity observed with the three isomers in the rat. However, they suggest that the differences in susceptibility to para-xylene observed between the rats and the guinea pigs might be due to toxicokinetic factors.     

Metabolism of styrene oxide in the rat and guinea pig

The metabolism of styrene oxide has been studied in the rat and guinea pig, with emphasis upon bivalent sulfur metabolites. Methylthio analogs of phenylethylene glycol, with the methylthio group in both possible positions, were found as urinary metabolites in both species. These compounds were present in more than trace amounts. The excretion of 2-hydroxy-1-methylthio-1-phenylethane amounted to about 7% of the administered dose in the guinea pig, and about 2% in the rat, in o-24 hr urine samples. The positional isomer 1-hydroxy-2-methylthio-1-phenylethane was excreted in lesser amounts in both species. Acidic urinary metabolites derived from glutathione conjugates are species dependent. In this study, the only products observed in the rat were the mercapturic acids expected as a result of reaction of the oxide with glutathione. In the guinea pig, the major bivalent sulfur acids were the corresponding mercaptoacetic acids. Other related metabolites included a mercaptolactic and a mercaptopyruvic acid, together with one of the mercapturic acids. These metabolites result from partial acetylation or acetylation/deacetylation of cysteine or cysteinylglycine adducts. The hitherto unobserved dihydrodiol formed via an arene oxide was found as a minor metabolite for both styrene and styrene oxide.     

2,3,7,8-tetrachlorodibenzo-p-dioxin and natural immunity: lack of an effect on the complement system in a guinea pig model

We examined the complement system as a potential target for toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in normal Hartley guinea pigs. Guinea pigs were injected intravenously with 0.5 microg/kg TCDD and bled at weekly intervals up to 1 month following injection. Serum samples were collected at each time point and assayed for total hemolytic complement activity (CH50), alternative pathway complement activity (AH50) and complement component C4 functional activity. Injection of TCDD led to a wasting syndrome, as observed by a lower body weight gain in TCDD-treated animals when compared to control animals. However, at this dose, TCDD failed to induce any significant change in complement activity as determined by all three methods used in this study. The results indicate that, at 0.5 microg/kg body weight, a dose slightly lower than the LD50 value in guinea pigs, TCDD fails to affect the complement system.     

The inhibition of rat and guinea pig cholinesterases by anionic hydrolysis products of methylphosphonic difluoride (difluoro)

Methylphosphonic difluoride (difluoro) and its hydrolysis products, methylphosphonofluoridate (MF) and fluoride, were examined for cholinesterase-inhibiting ability in rats and guinea pigs by both inhalation and intraperitoneal exposure routes. In vivo inhibition was compared to in vitro inhibition. In the whole animal, MF was the active chemical, but in vitro under special conditions, difluoro was more potent than MF and fluoride. Rat and guinea pig blood cholinesterase were equally sensitive to inhibition by MF, but only the guinea pig displayed cholinergic signs leading to death from MF toxicity. Data imply that MF is responsible for the cholinesterase inhibition resulting from exposure to DF vapor. MF may be the first example of a moderately strong acid shown to inhibit cholinesterase and cause death from cholinergic effects.     

Nickel chloride-induced metabolic changes in the rat and guinea pig

This study was undertaken to explore the toxic effects of nickel chloride (NiCl₂) on body metabolism and to elucidate the mechanism of action involved. Nickel chloride was given by various routes: intraperitoneal (8 mg Ni/kg), intratracheal (1 mg Ni), and by long-term ingestion (drinking water, 225 ppm). In addition, an intragastric [¹⁴C]glucose load (600 mg) was also given to some of the intratracheally injected animals. The following parameters were measured in the serum to assess the effect of Ni on metabolism: total ¹⁴C radioactivity, glucose, insulin, total lipids, cholesterol, and triglycerides. Liver glycogen and glucose-6-phosphatase activity was measured to determine glucose turnover in the liver. ⁶³Ni tissue distribution and excretion of ⁶³Ni, calcium, sodium, potassium, zinc, and glucose were also measured as a determination of metal and carbohydrate metabolism, A single intraperitoneal or intratracheal injection of Ni to rats caused a rapid transient increase in serum glucose, but a decrease in serum insulin and glucosuria. When exogenous insulin was given at the same time as the nickel challenge, the elevation of serum glucose was prevented. Glucose turnover studies indicated that the mechanism of action of nickel appears to be in the inhibition of insulin release. This inhibition of insulin release could be related to the extremely high concentration of nickel found in the pituitary and the effect of nickel on the secretion of the pituitary hormones (GH and ACTH).     

The low KM-phosphodiesterase inhibitor denbufylline enhances neuronal excitability in guinea pig hippocampus in vitro

Summary The actions of the phosphodiesterase inhibitor denbufylline on the excitability of hippocampal neurons were investigated by means of extracellular and intracellular recordings. Denbufylline, which has been shown to selectively inhibit a low K_M, Ca²⁺/calmodulin-independent phosphodiesterase isozyme, concentration-dependently increased the amplitude of the extracellularly recorded CAI population spike evoked by electrical stimulation of the Schaffer collateral/commissural pathway. Concentration-response-curves yielded an EC₅₀ for denbufylline of 0.76 M. In comparison, the nonselective phosphodiesterase inhibitor 3-isobutyl-lmethylxanthine (IBMX) also produced an increase in the amplitude of the population spike. From the concentration-response-curve, which was steeper than that of denbufylline, an EC₅₀ for IBMX of 1.04 M was obtained. However, despite their similar EC₅₀ values, denbufylline was found to be significantly more potent at lower concentrations (<- 300 nM) than IBMX.Intracellular recordings from CAI pyramidal cells revealed postsynaptic actions of denbufylline (300 nM) as indicated by a small drug-induced depolarization (2 – 5 mV) associated with an increase in membrane input resistance by 10–20%. In addition, denbufylline blocked the accommodation of trains of action potentials evoked by the injection of depolarizing current pulses.The results suggest i) that accumulation of adenosine-3,5-monophosphate (CAMP) in the postsynaptic cell and/or in the presynaptic terminal produced by blockade of phosphodiesterases leads to enhanced synaptic transmission in the CAI area of the hippocampus and ii) that a low K_M, Ca ²⁺/calmodulin-independent cAMP-phosphodiesterase is an important component involved in the regulation of the intracellular cAMP level at synapses of central nervous system neurons. Send offprint requests to B. Sutor at the above address     

Toluene toxicokinetics and metabolism parameters in the rat and guinea pig

Cochlear disruptions induced by toluene were shown in the rat but not in the guinea pig. To better understand the differences between species, three investigations were carried out to study (1) the blood affinity and the pulmonary uptake of the solvent, (2) its clearance and (3) its urinary elimination in both species. The blood affinity of toluene was +44% higher in the rat than in the guinea pig (14.4 μg/g versus 10 μg/g). Similarly, the pulmonary uptake of toluene was approximately 46.5% more efficient in the rat than in the guinea pig (75.4 μg/g versus 40.3 μg/g) after 3 h inhalation of 1500 ppm toluene. Therefore, the physicochemical composition of the blood could explain the difference in the uptake performances between rats and guinea pigs. The clearance of the toluene showed that 10 min after an intravenous administration of 400 μL of vehicle containing 28 μL (43 mg kg⁻¹) of toluene, the solvent concentration was approximately threefold higher in the rat than in the guinea pig blood. The last experiment was carried out to compare the concentrations of the urinary metabolites. The concentrations of o-cresol, hippuric and benzyl mercapturic acids measured in the urines were different before and after the toluene injection. These data give evidence for large differences of toluene uptake and metabolism between rat and guinea pig. Therefore, it seems reasonable to claim that guinea pigs cochleas are not susceptible to toluene as the blood burden of solvent does not reach the concentration required to induce permanent damages.     

Effects of Ginkgo biloba extract (EGb 761) on the guinea pig vestibular system.

Previous studies have demonstrated that the administration of Ginkgo biloba extract (EGb 761) improves the compensation of the vestibular syndrome induced by transection of the VIIIth nerve. To investigate the mechanisms at play, the vestibular nuclei of alert guinea pigs were perfused with EGb 761. This perfusion always induced a stereotyped reversible postural syndrome that was the mirror image of the syndrome provoked by the unilateral lesion of the otolithical receptors. This result supports the hypothesis that EGb 761 has a direct excitatory effect on the lateral vestibular nuclei (LVN) neurons. In a second step, we quantified the horizontal vestibuloocular reflex (HVOR) of the normal guinea pig following IP injection of EGb 761. In normal guinea pig, IP administration of EGb 761 led to a reversible, dose-dependent decrease of the HVOR gain without affecting the phase of the reflex. These data help to explain the therapeutic effects of EGb 761 during vestibular syndromes and strongly suggest an impact at the neuronal level.     

Ototoxicity of cis-dichlorodiammine platinum (II) in the guinea pig.

cis-Dichlorodiammine platinum (II) (NSC-119 875), an agent with potent antineoplastic activity which also induces renal, intestinal, and bone marrow toxicity, was tested for ototoxic effects in guinea pigs. Ototoxicity was evaluated by the disappearance of Preyer's reflex in response to pure tones of 5, 7, and 10 kHz and by histopathological evaluation of the inner ear with surface preparations or midmodiolar sections. Groups of five guinea pigs treated with 8–40 ip injections of cis-dichlorodiammine platinum (II) 1 mg/kg (5 doses/wk) or with 10–15 doses of 1.5 mg/kg developed permanent deafness and histopathological lesions with pronounced loss of outer hair cells in the lower turns of the organ of Corti. Frequently, entire rows of outer hair cells disappeared and only a few isolated hair cells remained intact. Moderate numbers of outer pillar cells were missing and occasional inner hair cells and pillar cells were also damaged. Hair cell lesions included degeneration, complete cytolysis, and replacement by phalangeal scars. Single doses of 6, 9, 12, or 18 mg/kg of cis-dichlorodiammine platinum (II) produced permanent hearing loss as early as Day 3 and a scattered pattern of outer hair cell loss on Day 4 with cytological changes similar to but less severe than those observed for multiple doses. Neomycin sulfate-treated guinea pigs used as positive controls (eight consecutive daily doses of 150–250 mg/kg) showed a similar pattern of hair cell loss as produced by cis-dichlorodiammine platinum (II). Positive controls treated with a single dose of 6-aminonicotinamide from 2.5 to 20 mg/kg, developed severe damage to both inner and outer hair cells, pillar cells, and spiral ganglia. Occasional strial atrophy was also observed.     

Linopirdine (DuP 996) selectively enhances acetylcholine release induced by high potassium,but not electrical stimulation,in rat brain slices and guinea pig ileum

The enhancement of acetylcholine (ACh) release during electrical or potassium depolarization was compared in the presence of either linopirdine or 4-aminopyridine (4-AP) using two different functional assays. We measured ACh and choline (Ch) directly from striatal perfusates using high performance liquid chromatography and electrochemical detection or indirectly by measuring ACh-mediated twitch height changes in the electrically stimulated guinea pig ileum. Potassium-induced twitch responses were also measured in resting ileal segments. Linopirdine significantly enhanced ACh release in the presence of elevated potassium levels in rat striatal slices and guinea pig ileum, but had no effect during electrical stimulation in either model. In contrast, 4-AP enhanced ACh release in both electrically stimulated striatal slices and ileal strips. In agreement with other studies, atropine enhanced ACh release during either electrical or potassium stimulation paradigms in the striatal slice. The in vitro observations in this report suggest that linopirdine has limited ability to augment the cholinergic neurotransmission evoked by electrical stimulation. © 1993 Wiley-Liss, Inc.     

The metabolism and disposition of fenofibrate in rat, guinea pig, and dog

The metabolism and disposition of orally administered single doses of [14C]fenofibrate (isopropyl 2-[4-(4-chlorobenzoyl)phenoxy]-2- methylpropionate) have been studied in rat, guinea pig, and dog. In rats, the urinary excretion of 14C in 5 days varied from 11 to 51% of the dose and was markedly dependent upon the dose form given. The interpretation of these data in terms of factors affecting the absorption of fenofibrate from the gut is complicated by the enterohepatic recirculation of metabolites. The tissue distribution of 14C after oral administration of an ethanolic solution of fenofibrate has been studied in the rat. The only tissues in which the concentration of 14C exceeded that in the blood were the organs of absorption and elimination, the gut, liver, and kidneys. Guinea pigs excreted 53% of the dose in the urine in 5 days, with a further 34% in the feces, while in dogs the corresponding figures were 9% and 81%, respectively. In all three species, all the urinary metabolites were products of ester hydrolysis, and the principal excretion product was "reduced fenofibric acid" which arose by subsequent carbonyl reduction. Glucuronidation of fenofibric acid and "reduced fenofibric acid" was a very minor reaction in the rat and guinea pig and was not detected in the dog. In addition, polar unknown metabolite(s) were detected in all three species, but were not investigated further. The results are discussed in terms of the comparative disposition of fenofibrate and other hypolipidemic agents and the contribution of these findings to the safety assessment of such drugs.     

Formation of methylthio metabolites of indene in the guinea pig and the rat

The formation of methylthio metabolites of epoxides has been shown to be a significant route of metabolism in some species. Several aspects of this metabolic conversion for indene were examined. Two isomers of hydroxy(methylthio)indane were found in the urine of guinea pigs administered indene (14.3 and 100 mg/kg, ip). The major isomer, 2-hydroxy-1-methylthioindane (I) was present as 6-9% of the administered dose after 24 hr, while lower amounts (0-0.6%) of a minor isomer (II) were observed. A significant amount of isomer I was found as a urinary metabolite of indene oxide (14% of 12.5 mg/kg, ip). To further elucidate the route of formation of I, the glutathione (I-GLU) and mercapturic acid (I-MER) conjugates of indene oxide were synthesized and administered to the guinea pig. The methylthio metabolite I was present as a significant urinary metabolite of both conjugates of indene oxide, comprising 9.6% and 5.7% of the dose of I-GLU (5 mg, ip) and I-MER (4 mg, ip), respectively. These results show that the formation of a hydroxy(methylthio)indane is a significant route of metabolism for indene and indene oxide in the guinea pig, and that this metabolite arises via further metabolism of conjugates in the glutathione pathway. In the rat, isomer I is a minor metabolite. Mechanistic aspects of the formation of these thioether metabolites are discussed.     

Protective effect of O-phenanthroline against mechlorethamine toxicity in the rat liver slice system and in the guinea pig skin

The effect of O-phenanthroline (OP) on mechlorethamine hydrochloride (HN2) toxicity was studied in in vitro and in vivo experiments. Incubation of HN2 with the in vitro rat liver slice system resulted in leakage of alanine aminotransferase (ALT) in a time-dependent manner. Exposure of the slices to HN2 for 4 h caused 79.2% ALT leakage. In the presence of OP enzyme leakage was reduced to 28.7%. OP-induced protection was shown to be dose dependent. Other metal chelators such as dithiothreitol (DTT) and EDTA (ethylenediaminetetraacetic acid) had a weak effect on HN2 cytotoxicity. The protective activity of OP was also demonstrated in in vivo skin toxicity studies in the guinea pig. The ulcerative effect of topically applied HN2 was inhibited by OP even when applied 10 min following the alkylator. Histology of NH2-treated skin showed epidermal ulceration associated with a covering layer of encrusted exudate. However, only a slight diffuse acanthosis of the epidermal layer was observed when OP was applied 10 min after the vesicant. It is suggested that OP may be used for the prevention of tissue damage caused by antineoplastic treatment with nitrogen mustard. It might also be employed in military medicine as an antidote to the chemical weapon sulfur mustard.     

In vivo studies on the role of complement in the clearance of liposomes in rats and guinea pigs.

The ability of complement (C) system to remove liposomes from blood circulation was examined in vivo using rat and guinea pig as models. Although the liposomes were not degraded in guinea pig serum in vitro, they were degraded remarkably in guinea pig circulation, as assessed by the urinary excretion of [3H]inulin released from liposomes. The suppression of rat C system to 64% normal C hemolytic activity by treating animals with K76COOH agent resulted in a significant decrease in both the uptake of liposomes by liver and the release of [3H]inulin, providing in vivo evidence for C-mediated clearance of liposomes in rats via uptake by macrophages and degradation in blood circulation, respectively. On the other hand, the K76COOH-induced suppression of C (70% normal hemolytic activity) in guinea pigs slightly increased both the hepatic uptake and the release of [3H]inulin. In addition, the hepatic uptake and in vivo degradation in guinea pigs varied in an opposite manner when the animals were preloaded by empty liposomes or when the liposome size and cholesterol content varied. These results suggest there is a difference between the factors involved in liposome degradation and the factors involved in hepatic uptake and also support the likelihood that there is no C-mediated degradation in guinea pigs.