Examination of model enzyme and penetration systems in relation to antibacterial activity

Recent work has shown that the activity of cephalosporins in inhibiting exocellular DD-peptidases from Streptomyces R61 and Actinomadura R39 are, at best, only poorly related to minimum inhibitory concentrations against pathogenic isolates. Taking into account the rate at which cephalosporins diffuse through porin channels, such as exist in certain Gram-negative organisms, does not help in establishing a relationship between MIC data and the kinetic data on the model enzymes. Most published cell wall permeability studies, the porin ones being a principal exception, have not examined long enough series of structurally related compounds to establish property-activity relationships.     

Metabolism of stilbene estrogens and steroidal estrogens in relation to carcinogenicity

The oxidative metabolism of diethylstilbestrol (DES) and 17-ethynyl estradiol, as examples of stilbene- and steroid-type estrogens, is discussed with respect to the formation of reactive intermediates. For DES, a genotoxic potential is implied by metabolic studies and positive effects in short-term tests for genetic damage. A particularly important pathway for DES carcinogenicity appears to be peroxidase-mediated oxidation. Although data for steroidal estrogens are more ambiguous, the available evidence suggests that metabolic activation by peroxidatic oxidation may also be of importance for this class of estrogens.Abbreviations DES diethylstilbestrol, 3,4-bis-(p-hydroxyphenyl)hex-3-ene - E-DES trans-diethylstilbestrol - Z-DES cis-diethylstilbestrol - DIES dienestrol, 3,4-bis-(p-hydroxyphenyl)-hexa-2,4-diene (nomenclature of DES metabolites according to the system of Metzler and McLachlan 1978a) - E₁ estrone - E₂ estradiol-17ß - EE₂ 17-ethynylestradiol - 7,8-BF 7,8-benzoflavone - GC gas chromatography - HPLC high performance liquid chromatography - MS mass spectrometry Paper presented at the Satellite Symposium of the 24th Congress of the European Society of Toxicology, Rome, March 29, 1983     

Inhaled formaldehyde: evaluation of sensory irritation in relation to carcinogenicity

OBJECTIVES: The critical health effects of formaldehyde exposure include sensory irritation and the potential to induce tumours in the upper respiratory tract. In literature, a concentration as low as 0.24 ppm has been reported to be irritating to the respiratory tract in humans. Nasal tumour-inducing levels in experimental animals seem to be 1-2 orders of magnitude larger. In this paper, the subjectively measured sensory irritation threshold levels in humans are discussed in line with findings obtained in animal experiments. In addition, a Benchmark dose (BMD) analysis of sensory irritation was used to estimate response incidences at different formaldehyde concentrations. METHODS: Data on respiratory irritation and carcinogenicity of formaldehyde were retrieved from public literature and discussed. BMD analysis was carried out on human volunteer studies using the US-EPA BMD software. RESULTS: Subjective measures of irritation were the major data found in humans to examine sensory (eye and nasal) irritation; only one study reported objectively measured eye irritation. On a normalized scale, mild/slight eye irritation was observed at levels 1 ppm, and mild/slight respiratory tract irritation at levels 2 ppm. With the BMD software, it was estimated that at a level of 1 ppm, only 9.5% of healthy volunteers experience 'moderate' (i.e., annoying) eye irritation (95% upper confidence limit). An important factor modulating the reported levels of irritation and health symptoms most probably includes the perception of odour intensity. In several studies, the 0-ppm control condition was missing. From the results of the long-term inhalation toxicity studies in experimental animals, a level of 1 ppm formaldehyde has been considered a NOAEL for nasal injury. CONCLUSIONS: Sensory irritation is first observed at levels of 1 ppm and higher. From both human and animal studies, it was concluded that at airborne levels for which the prevalence of sensory irritation is minimal both in incidence and degree (i.e., <1 ppm), risks of respiratory tract cancer are considered to be negligibly low.     

Enzyme kinetics in relation to enzyme inhibitors

A Large number of pharmacologically important compounds have been found to act as enzyme inhibitors and a kinetic study of the inhibitory process can provide important information on the potency of the compound and on its mode of interaction with the enzyme. Kinetic studies are generally simple to perform and interpret but their misuse can easily result in erroneous and misleading conclusions. The purpose of this paper is to emphasize the points of kinetic theory that are most frequently misapplied. Since any study of the kinetics of enzyme inhibition will necessarily involve a study of the kinetics of the enzyme in the absence of inhibitor, the first part of this paper deals with such systems. The material discussed in this paper is in no way original and neither does it represent a complete survey of the kinetics of simple enzyme systems; such treatments may be found in a number of textbooks.^1–4 I have chosen to take specific cases in which the enzyme kinetic studies have been frequently misapplied and to illustrate these with reference to the simplest single substrate reaction system. I do not intend to list or give examples of specific errors that have appeared in the literature since the purpose of this paper is not to criticize but to provide information which I hope will lead to a more constructive use of enzyme kinetic studies.     

MPTP toxicity in relation to age, dopamine uptake and MAO-B activity in two rodent species

The effects of single and multidose 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) treatment on mice (NMRI and C-57bl) and rats of different ages have been investigated by using the reduction of striatal dopamine uptake rate as a measure of the neurotoxic effect. The possibility that differences in MAO-B activity or in dopamine (DA) uptake rate might explain differences in MPTP toxicity between species, strains or animals of different age was investigated. Single dose MPTP treatment (45 mg/kg) had no effect on 10 day and 3 week old mice, while there was a significant reduction of DA uptake rate at the age of 12 and 40 weeks (both strains). No neurotoxicity of single dose MPTP treatment was observed in the rats, irrespective of their age. Multidose treatment with MPTP (3 x 20-45 mg/kg) caused a reduction of DA uptake rates both in the mice and in the rats at all dose regimens used. The effect of MPTP increased with increasing doses and was most pronounced in the C-57bl mice. Both DA uptake rate and monoamine oxidase B (MAO-B) activity increased with age. The increase in MAO-B activity was highest between 10 days and 3 weeks both in the mice and in the rats. Rats had higher (MAO-B) activity than the mice, while the two species had about the same DA uptake rates. No obvious correlations were found either between MAO-B activities or DA uptake rates and the neurotoxic effect of MPTP.     

Hepatic peroxisomal and microsomal enzyme induction by citral and linalool in rats

The short-term effects of oral administration of citral and linalool to rats have been compared. Male Wistar rats were given, by gastric intubation, 1.5 g citral or linalool/kg body weight/day for 5 days. Citral caused peroxisome proliferation as indicated by induction of cyanide-insensitive palmitoyl-CoA oxidation and bifunctional enzyme; levels of microsomal cytochrome P-450 IVA1 were also raised. Linalool caused induction of the peroxisomal enzymes but not of cytochrome P-450 IVA1, indicating that it possesses activity somewhat different from that of citral. These results suggest that the mechanisms of peroxisome proliferation may be independent of induction of cytochrome P-450 IVA1.     

Trace elements and electrolytes homeostasis and their relation to antioxidant enzyme activity in brain hyperexcitability of epileptic patients

Epileptogenesis is a big challenge. Various experimental and human studies suggested that the homeostasis of trace elements, electrolytes, membrane lipid peroxidation, and antioxidants is crucial for brain function, and they were directly or indirectly implicated as taking part in the pathophysiology of neuronal excitability, neuronal excitotoxicity, and seizure recurrence and its resistance to treatment with antiepileptic drugs (AEDs). In addition, AEDs can also alter the homeostasis of trace elements, electrolytes, and seriously increase membrane lipid peroxidation at the expense of protective antioxidants, leading to an increase in seizure recurrence and an idiosyncratic drug effect. Differential effects were detected among different AEDs treatments in which carbamazepine (CBZ) was found to be better anticonvulsant for the control of free radical related seizures and the level of trace elements were better regulated with CBZ than with valproate (VPA) and phenytoin (PHT) therapies. It is concluded that adequate trace elements and antioxidants supply is important for brain functions and prevention of neurological diseases and further elucidation of the pathological actions of such substances in the brain should result in new therapeutic approaches. Trace elements and antioxidant might have neuroprotective biological targeted benefits when used in epileptic patients.     

Comparison of acute cardiovascular effects of cadmium and captopril in relation to oxidant and angiotensin converting enzyme activity in rats

Cadmium (0.1, 0.32, 1.0 mg/kg i.v.) produced dose dependent hypertensive response in pentobarbitone anesthetized Sprague-Dawley (S-D) rats. The peak hypertensive effect was observed within 15 min of cadmium administration. This response gradually decreased over 1-h observation period. Heart rate did not change significantly. Serum malondialdehyde (MDA) levels increased with cadmium administration. The lower dose of cadmium (0.1 mg/kg i.v.) increased serum MDA to 0.14 +/- 0.02 nmol/mL as compared to 0.12 +/- 0.01 nmol/mL in the control group and was not statistically significant. However, mid (0.32 mg/kg i.v.) and high doses (1.0 mg/kg i.v.) raised serum MDA levels significantly (P < 0.01). Cadmium inhibited serum angiotensin converting enzyme (ACE) levels at all the three tested doses and was statistically significant (P < 0.01). Captopril (1.0, 3.2 mg/kg i.v.) produced a dose dependent mild hypotensive response. The peak effect was observed within 5 min. Cadmium produced inhibition of serum ACE levels, however, a dose response effect was not observed. Captopril (3.2 mg/kg i.v.) decreased serum ACE levels to 5.4 +/- 1.1 U/mL (control levels 10.7 +/- 1.4 U/mL). Serum MDA levels were decreased by captopril treatment. A correlation between serum ACE and MDA following higher dose of cadmium was found. These results indicate that acute administration of cadmium, an inorganic blocker of ACE and calcium channels cadmium produced hypertensive response while captopril produced mild hypotensive response in rats.     

Inhibition of angiotensin converting enzyme activity by five Senecio species

In our continuous search of biological properties of Senecio species (Compositae), we investigated S. ambiguus subsp. ambiguus (Biv.) DC, S. gibbosus subsp. gibbosus DC, S. leucanthemifolius Poiret, S. inaequidens DC, and S. vulgaris L. for their angiotensin converting enzyme (ACE) inhibitory activity through an in vitro bioassay based on the enzymatic cleavage of the chromophore-fluorophore labelled substrate dansyltriglycine into dansylglycine, which is quantitatively measured by HPLC. Among analyzed extracts, ethyl acetate demonstrated the highest activity with IC₅₀ values of 192.1 and 219.1?μg/mL for S. ambiguus subsp. ambiguus and S. inaequidens, respectively. Flavonoids were detected in these extracts on TLC sprayed with Natural Products reagent - polyethylene glycal reagent (NP/PEG).     

Effect of cadmium on phenobarbital and polycyclic hydrocarbon-induced alterations in hepatic microsomal monooxygenase activity in the rat

The heavy metal, cadmium, is a potent inhibitor of the hepatic microsomal monooxygenase enzyme system in male, but not female, rats. The selectivity of this inhibitory effect of Cd was further examined by utilizing rats treated with inducers of this drug metabolizing enzyme system. Animals received phenobarbital (PB) sodium (100 mg/kg, ip, 72, 48, and 24 hr before sacrifice), or 3-methylcholanthrene or benzo[a]pyrene (20 mg/kg, ip, 72 and 48 hr prior to sacrifice) as inducers. Designated groups of these animals also received cadmium (1.0 mg Cd²⁺/kg), ip) either 120 or 72 hr before sacrifice. Noninduced male rats exhibited significant decreases in cytochrome P-450 content and drug-metabolizing enzyme activity following Cd treatment. The magnitude of the reductions in drug-metabolizing enzyme activity produced by Cd paralleled the magnitude of the sex difference in biotransformation of the substrate examined. Phenobarbital-treated male rats receiving a simultaneous Cd injection (72 hr prior to sacrifice) were also sensitive to Cd-induced inhibitions in cytochrome P-450 content and monooxygenase activity, although the extent of the reductions produced by Cd in PB-treated animals was less than that observed in noninduced male rats. In PB-treated male rats receiving a prior dose of Cd (120 hr before sacrifice), only the metabolism of the highly sex-dependent substrate, ethylmorphine, was significantly reduced. Cytochrome P-448 levels, and cytochrome P-448-mediated biotransformations which are elevated following hydrocarbon treatment, were not decreased by either simultaneous or prior Cd administration to male rats. Control, PB-treated, and hydrocarbon-treated female rats were not susceptible to Cd-induced reduction in hemoprotein content or inhibition of drug-metabolizing enzyme activity with the exception of those animals receiving both benzo[a]pyrene and Cd, which displayed slight but significant reductions in the oxidation of sex-dependent substrates. These results demonstrate the selective nature of the inhibitory effects of Cd upon drug metabolism in the rat.     

Diethylstilbestrol metabolic transformation in relation to organ specific tumor manifestation.

Oxidative biotransformation of the synthetic estrogen diethylstilbestrol (DES) gives rise to several reactive compounds. Two mechanisms are proposed concerning the possible involvement of reactive metabolites in the organotropic tumorigenesis of DES. The first mechanism suggests an affinity of the metabolite to the estradiol receptor present in estrogen target organs. This has been shown for the olefinic epoxide of DES. The second mechanism is based on the organ specific oxidation of DES by peroxidase. The intermediates of this reaction were found to bind to nucleic acid and protein in a manner characteristic of chemical carcinogens.     

In vitro kinetics of coumarin 3,4-epoxidation: application to species differences in toxicity and carcinogenicity.

Coumarin, a natural product and fragrance ingredient, is a well recognized rat liver toxicant, and dietary administration at toxic dosages increased the incidence of rat cholangiocarcinomas and parenchymal liver-cell tumors in a chronic bioassay. Hepatotoxicity in rats is site- and species-specific, and is thought to result from the formation of coumarin 3,4-epoxide and its rearrangement product, o-hydroxyphenylacetaldehyde (o-HPA). The goals of the current study were to describe the in vitro kinetics of the metabolic activation of coumarin, and determine whether species differences in susceptibility to liver injury correlate with coumarin bioactivation determined in vitro. Coumarin 3,4-epoxidation was quantified via the formation of o-HPA in pooled hepatic microsomes from female B6C3F1 mice, male F344 rats, and individual humans (n = 12 subjects), and the apparent kinetic constants for o-HPA production were calculated using nonlinear regression and fitting to either a one-enzyme or two-enzyme model. Eadie-Hofstee analyses indicated that o-HPA formation was biphasic in both rat and mouse liver. Although the apparent high affinity K:(m) in rat and mouse liver microsomes was 38.9 and 47.2 microM, respectively, the overall rate of o-HPA formation was far greater in mouse than in rat liver microsomes. Furthermore, the total clearance (CL(int)) of coumarin via o-HPA formation in mouse liver microsomes was 4-fold greater than in rat liver microsomes. Since mice are relatively resistant to hepatotoxicity, the data indicated that rates of o-HPA formation in rat and mouse liver microsomes were not directly predictive of liver toxicity in vivo, and further suggested that o-HPA detoxification played a role in modulating coumarin-mediated toxicity. The current studies also indicated that coumarin 3,4-epoxidation in human hepatic microsomes was minimal. In human liver microsomes (n = 12), the kinetics of o-HPA formation were best described by a single enzyme model, with the K(m) for o-HPA formation ranging from 1320-7420 microM. In the most active human sample, the intrinsic clearance of coumarin via the 3,4-epoxidation pathway was 1/9 and 1/38 that of the rat and mouse, respectively. The in vitro kinetics of o-HPA formation, and in particular, the large quantities of coumarin required for o-HPA production in human liver microsomes, strongly suggest that humans are unlikely to produce toxicologically relevant concentrations of this metabolite following low level coumarin exposures.     

Highly toxic coplanar PCB126 reduces liver peroxisomal enzyme activities in rats

The effect of the highly toxic coplanar PCB congener, 3,4,5,3',4'-pentachlorobiphenyl (PCB126) on hepatic peroxisomes was studied in rats. The aim of this study was to investigate whether a toxic dose of the dioxin-like coplanar PCB modifies enzyme activities in peroxisomes where plays an important role in lipid metabolism. Treatment with PCB126, at a single i.p. administration of 25 mg/kg which evokes clear suppression of body weight gain, resulted in marked reduction (to about 40-50%) of catalase activity and peroxisomal fatty acyl-CoA β-oxidizing system. Immunoblotting showed that expression of catalase was greatly reduced by the treatment in parallel with the activity. Light microscopy revealed a drastic reduction in granules possessing peroxidase activity, while electron microscopy demonstrated that no apparent morphological changes had taken place. Thus the reduction in catalase activity caused by PCB126 could be attributable to suppression of protein expression. The marked reduction of these peroxisomal enzyme activities might be related to hyperlipidemia caused by dioxin-related compounds in rats and humans.     

Involvement of the extracellular signal regulated kinase pathway in hydrocarbon-induced reactive oxygen species formation in human neutrophil granulocytes

In the present study we have examined the effects of hydrocarbons on the formation of reactive oxygen species (ROS) in human neutrophil granulocytes in vitro. We found that hydrocarbons induce ROS formation in a concentration-dependent manner and that the ROS-inducing potency increases with increasing number of carbon atoms in the structure. In general, aromatic hydrocarbons were less potent inducers of ROS than aliphatic and cyclic hydrocarbons. The most potent compound in each group, t-butylcyclohexane, n-decane, and n-butylbenzene, were chosen for mechanistic studies. ROS formation was inhibited by the MEK1/2 inhibitor U0126, the tyrosine kinase inhibitor erbstatin-A, and the phosphatidylinositol-3 kinase inhibitor wortmannin. The involvement of the ERK1/2 pathway was confirmed by Western blot analysis of phosphorylated ERK1/2. The study revealed only small differences in the mechanisms involved for the three compounds. The responses were not affected by Pertussis toxin, indicating that Gi-protein coupled receptors are not involved in neutrophil activation after hydrocarbon exposure. Based on these findings we propose a mechanism involving tyrosine kinases, PI3 kinase, and the ERK1/2 pathway, leading to activation of the NADPH oxidase and production of ROS in neutrophils stimulated by organic solvents.     

Ultrastructure of Mycoplasma pneumoniae exposed to macrolide antibiotics during cultivation on a glass surface and in an organ culture, in relation to their mycoplasma-killing activity

The macrolide antibiotics midecamycin acetate (MOM), erythromycin (EM), midecamycin (MDM), josamycin (JM) and rokitamycin (RKM) showed killing activity against Mycoplasma pneumoniae strain FH-P24. The activity of MOM, EM and JM was not influenced by the number of organisms inoculated, but that of RKM was markedly decreased by a large inoculum. Scanning electron microscopic observations showed many long filaments crossing over each other with small colonies when the organisms were cultivated on a glass surface without any drug for 72 h. When they were exposed to four times the MIC of MOM, the filamentous forms were decreased and the colonies did not grow to a large size. However, after exposure to the other macrolides, the colonies grew larger. Transmission electron micrographs revealed that intracellular vacuolization of the organisms was induced by exposure to MOM, EM and JM, but a mixture of vacuolized cells and "young" organisms was observed after exposure to MDM and RKM. In hamster tracheal organ cultures, the number of organisms was greatly decreased by exposure to four times the MIC of MOM, and the remaining filamentous and colonized organisms were lysed. However, treatment with four times the MIC of the other macrolides induced hardly any lysis of the organisms. In transmission electron micrographs, filamentous and rounded organisms filling the ribosome-like matrix could be seen on the epithelial surface. Treatment with four times the MIC of these macrolides decreased the number of organisms and vacuolized filamentous organisms could be seen on the epithelial cells.