Rapid absorption from the urinary bladder of a series of n-alkyl carbamates: a route for the recirculation of drugs.

1 The rate of loss of a series of n-alkyl carbamates from the lumen of the urinary bladders of female rats has been studied. 2 The rate of loss obeys first order kinetics and was not affected by water flux across the bladder wall nor by binding of the carbamates to it. 3 The rate of loss of octyl carbamate was reduced by about 76% by the presence of 5% Tween 80. Histological evidence indicates that this may be due to the formation of a thin luminal lining which may be adsorbed Tween 80 or mucopolysaccharide material. 4 The absorption rate of the carbamates was limited by their hydrophilicity but reached a plateau for the more lipophilic homologues with a half life for loss of approximately 10 min. 5 The implications of these results with regard to the recirculation of unmetabolized drugs and hydrolysed conjugates of drugs, the systemic absorption of intravesically applied cancer chemotherapeutic agents and bladder wall permeability to carcinogens are discussed.

A novel role for carboxylesterase in the elevation of cellular cysteine by esters of cysteine.

Esters of cysteine, such as cysteine isopropylester (CIPE) or cysteine cyclohexylester (CCHE), are efficient delivery systems for cysteine to cells. After enzymic cleavage, the esters of cysteine provide a source of cellular cysteine, which may support reduced glutathione (GSH) synthesis and/or act as a direct chemoprotectant. Reducing esterase activity of rat lung slices or isolated hepatocytes with paraoxon or bis(4-nitrophenyl) phosphate or by reducing the temperature to 4 degrees dramatically altered the metabolism of esters of cysteine; the initial increase in cellular cysteine was slowed, the residency time of cysteine esters in the extracellular pool was prolonged without substantially enhancing the levels of intracellular ester. Incubation of lung slices with CIPE at 4 degrees led to a marked increase in cellular cysteine, which prior inhibition of esterase activity abolished. Inhibiting the neutral amino acid uptake systems, ASC and L, while effecting the uptake of cysteine, did not reduce the elevation of cellular cysteine by CIPE. We propose that the elevation of cellular cysteine by esters of cysteine may be mediated by membrane associated esterase activity.

Structure-activity relationships of cysteine esters and their effects on thiol levels in rat lung in vitro.

Pretreatment with cysteine esters increases cysteine (CySH) levels in rat lung and protects against the lethal effects of inhaled perfluoroisobutene in vivo. There are marked differences in the duration of protection achieved with different cysteine esters. In this study we have compared the uptake and metabolism of CySH, N-acetyl cysteine (NAc), cysteine esters and cystine esters in vitro using rat lung and liver homogenates and lung slices. Liver homogenates metabolized CySH and cysteine esters faster than lung homogenates. The half life (T1/2) of CySH in lung was 58.8 +/- 17.3 min and in liver was 14.0 +/- 1.6 min (mean +/- SEM). T1/2 of the esters in lung ranged between 6.5 and 12.1 min and in liver between 1.9 and 5.3 min. Cysteine tertiary butyl ester, which does not protect in vivo, was not hydrolysed to CySH by lung or liver homogenates. All esters increased and prolonged intracellular CySH concentrations in lung slices to a much greater extent than CySH itself. NAc did not raise intracellular CySH above that of the controls and no NAc appeared within the slice. After CySH incubation intracellular CySH was 0.9 +/- 0.1 nmol/mg wet wt at 10 min whereas after incubation with the esters it ranged between 2.60 and 3.65 nmol/mg wet wt. Cysteine cyclohexyl ester prolonged the increase of CySH the longest and cysteine methyl ester the shortest. CySH levels with cysteine cyclohexyl ester were 2.74 +/- 0.15 and 4.13 +/- 0.37 nmol/mg wet wt at 10 and 60 min, respectively, whereas with cysteine methyl ester, CySH levels were 2.60 +/- 0.5 and 1.25 +/- 0.08 nmol/mg wet wt at similar times. Cystine esters increased intracellular concentrations of both cystine and CySH. CySH concentrations ranged between 2.92 and 3.19 nmol/mg wet wt and cystine between 1.39 and 1.47 nmol/mg wet wt at 60 min. The elevation and duration of CySH in lung slices is well correlated with the duration of protection against perfluoroisobutene achieved in vivo.

Elevation of cysteine and replenishment of glutathione in rat lung slices by cysteine isopropylester and other cysteine precursors.

In this study, we have used a rat lung slice model to compare the ability to several potential cysteine delivery systems (L-cysteine isopropylester, L-cysteine cyclohexylester, N-acetylcysteine, L,2-oxo-4-thiazolidine carboxylic acid and cysteine) to elevate cysteine and glutathione (GSH) levels in control lung slices and slices depleted of their GSH by diethyl maleate. The esters of cysteine produced the greatest rise in lung slice cysteine. All the cysteine delivery systems were capable of replenishing GSH in lung slices previously depleted of GSH by diethyl maleate.

Cysteine isopropylester protects against paracetamol-induced toxicity.

Cysteine isopropylester (CIPE), a novel ester of cysteine, has been synthesized in order to evaluate its potential as a chemoprotectant. The increased lipophilicity of the ester relative to cysteine should facilitate its entry into cells where, following hydrolysis, it should act as an intracellular source of cysteine or be utilized for the synthesis of glutathione so protecting the cell against various types of chemical insult. In this study, we evaluate the ability of CIPE to protect against paracetamol-induced hepatotoxicity in mice. When administered to mice, CIPE produced a rapid but transient elevation of levels of non-protein sulphydryls (NPSH) in liver, lung, kidney and spleen. The greatest increase in NPSH was seen in the lung, but after 60 min all NPSH values had returned to control levels, demonstrating the capacity of the mouse to rapidly metabolize both CIPE and cysteine. In mice pretreated with benzo(a)pyrene, CIPE protected against paracetamol-induced toxicity as measured by the prevention of mortality, the fall in hepatic NPSH and the decreased elevation of serum transaminases. CIPE (1.5 mmol/kg) appeared as effective as N-acetylcysteine (1.5 mmol/kg). Higher doses of CIPE (3.0 mmol/kg) alone were toxic to mice induced with benzo(a)pyrene but not to control or phenobarbitone-induced mice. The mechanism of this increased toxicity is unclear. CIPE has a short in vivo half life but was capable of protecting against paracetamol-induced toxicity. The potential of CIPE and other related cysteine esters to act as chemoprotectants merits further investigation.

The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes.

The binding constants of an homologous series of n-alkyl (C2-C10) carbamates (see formula in text) to the cytochrome P-450 of suspensions of isolated, viable rat hepatocytes have been measured. All the carbamates except ethyl and propyl carbamate produced type I difference spectra and their binding affinities (1/Ks) were found to be directly dependent upon their lipophilicity. These binding affinities were similar to those determined in rat liver microsomes. Maximum development of the binding spectrum in hepatocytes was always within one second of the addition of each carbamate, indicating that for these carbamates membrane permeability was not rate limiting for access to, and metabolism by, cytochrome P-450 and that much of the cells' cytochrome P-450 was unoccupied by endogenous substrates. Th major metabolites of C4-C8 carbamates were unconjugated omega-1 oxidation products. Below hexyl carbamate only the omega-1 hydroxylated metabolite was observed but for the more lipophilic carbamates the keto metabolite was also a major product. The same products were found in blood after i.p. dosing of rats with hexyl carbamate. A direct relationship was observed between the affinity constant of the carbamate for cytochrome P-450 and the total rate of oxidative metabolism in the omega-1 position. Hydrolysis of the carbamate group was a minor metabolic pathway in contrast to the in vivo situation.

Protection of rats against the effects of alpha-naphthylthiourea (ANTU) by elevation of non-protein sulphydryl levels.

We have investigated the influence of the elevation of pulmonary glutathione (GSH) levels on the toxicity of the rodenticide alpha-naphthylthiourea (ANTU) to rat lung. Administration of phorone (diisopropylidene acetone; 200 mg/kg i.p.) caused an initial depletion of both pulmonary and hepatic GSH followed after 48 hr by a marked elevation in both tissues, due most probably to a compensatory rebound synthesis. In control rats, ANTU produced a dose-dependent lethality, hydrothorax and loss of ability of lung tissue to accumulate adenosine and spermidine (markers of endothelial and epithelial cell function, respectively). These effects were prevented or markedly ameliorated when ANTU was given 48 hr after pretreatment with phorone. The mechanism of the protection by phorone pretreatment against ANTU-induced pulmonary toxicity is unclear. It may be due, in part, to elevated GSH levels in pulmonary endothelial cells and, in addition, to increased detoxification of ANTU in the liver, resulting in a decreased availability to the lung.

Protection by cysteine esters against chemically induced pulmonary oedema.

Perfluoroisobutene (PFIB) is a hydrophobic reactive gas produced by the pyrolysis of polytetrafluoroethane which induces pulmonary oedema similar to that induced by phosgene when inhaled. When a lethal dose is inhaled by Porton strain rats total non-protein thiol (NPSH) and glutathione (GSH) in the lung are reduced by between 30 and 49%, respectively. If the endogenous levels of thiols in the lung are reduced by pretreatment with buthionine sulfoximine (BSO) 16 hr before exposure to PFIB, the rats become more susceptible to the effects of the gas. The effect of BSO pretreatment on toxicity was prevented by pretreatment 30 min before exposure, with 5 mmol/kg N-acetylcysteine (NAc). NAc increased the levels of cysteine (CySH) in the lung by 150% and GSH was unaffected. Similarly pretreatment with 3 mmol/kg CySH also protected against toxicity and raised CySH levels by 100%. A series of cysteine esters and cystine dimethyl ester (CDME) have been synthesised which selectively raise lung levels of CySH in the rat lungs after intraperitoneal (i.p.) injection. The methyl ester and CDME raised lung levels of CySH by 4000 and 2000%, respectively, 10 min after i.p. injection whilst GSH levels remained unchanged. Cysteine isopropyl ester raised lung levels of CySH by 10,600% but liver levels by only 1400%. All esters except the t-butyl ester (CTBE) also raised maximal plasma levels of NPSH by up to 500%; however, when NAc was injected plasma levels increased by over 1500%. Rats treated with these esters at 3 mmol/kg and with NAc at 5 mmol/kg were protected against lethal doses of PFIB in all cases except when CTBE was used. It appears that these cysteine esters may distribute preferentially into the lung, unlike NAc. The selective enhancement of pulmonary CySH levels may provide a method for the protection of lungs against inhaled reactive toxicants by increasing intracellular CySH. Levels of CySH may also be raised in epithelial lining fluid thus reducing access of gaseous toxicants to pulmonary tissue.

Elevation of endogenous nucleophiles in rat lung by cysteine and glutathione esters in vitro.

In this study, we have compared the uptake of L-cysteine (L-CySH), D-cysteine (D-CySH), L-cysteine isopropyl ester (L-CIPE) and D-cysteine isopropyl ester (D-CIPE) in rat lung slices and tracheal sections and determined the effectiveness of glutathione (GSH), GSH isopropyl monoester, GSH isopropyl diester, gamma-glutamylcysteine (gamma-glu-cys) isopropyl monoester and gamma-glu-cys isopropyl diester to elevate and prolong intracellular GSH concentrations in rat lung slices. Lung slices were incubated with 1.0 mM of thiol and the concentrations determined intracellularly and extracellularly with time. Slices incubated with GSH, GSH isopropyl diester and gamma-glu-cys isopropyl diester had cellular GSH concentrations increased by up to 60%, 95% and 58%, respectively, whereas GSH isopropyl monoester and gamma-glu-cys isopropyl monoester did not increase the intracellular GSH concentration. Extracellularly, the GSH concentration had decreased by 15%, GSH isopropyl diester by 27%, gamma-glu-cys isopropyl diester by 66% and both isopropyl monoesters by over 90% at 120 min. Lung slices and tracheal sections incubated with L- or D-CySH at 37 degrees had increased cellular concentrations of L- and D-CySH which ranged between 0.88-1.25 nmol mg(-1) and 1.35-2.25 nmol mg(-1) , respectively. Reducing the incubation temperature to 4 degrees had little effect on the accumulation of D-CySH; however, L-CySH concentrations increased progressively in the trachea and lung to reach 2.73 and 2.63 nmol mg(-1) at 90 min, respectively. Lung slices incubated with L- or D-CIPE had increased L- or D-CySH concentrations up to a max of 13.7 and 11.1 nmol mg(-1) and tracheal sections up to a max of 5.56 and 11.09 nmol mg(-1). In the lung slice medium, L- and D-CIPE levels had decreased by 75.2% and 74.0% at 90 min, respectively, and from the tracheal section medium, L- and D-CIPE concentrations had decreased by 66.7% and 32.7%, respectively. Preincubation of lung slices and tracheal sections with the carboxylesterase inhibitor, bis (p-nitrophenyl) phosphate (BNPP), almost completely prevented the disappearance of L- and D-CIPE extracellularly and greatly reduced the appearance of cellular L- and D-CySH. GSH, GSH isopropyl diester and gamma-glu-cys isopropyl diester elevated and prolonged GSH concentrations in rat lung slices, but GSH isopropyl monoester and gamma-glu-cys isopropyl monoester did not increase GSH levels. The uptake of L-CySH, but not D-CySH, is temperature sensitive in rat lung slices and tracheal sections and carboxylesterases appear to have a major influence on the uptake and metabolism of L- and D-CIPE by rat lung slices and tracheal sections.

The effects of drugs, other foreign compounds, and cigarette smoke on the synthesis of protein by lung slices.

The incorporation of 14C-leucine into rabbit lung slices was monitored in the absence and presence of selected drugs and chemicals relevant to the perturbation of lung function and the development of lung disease. Known inhibitors of protein synthesis (cycloheximide and ricin) inhibited the incorporation of 14C-leucine. Marked inhibition was also recorded with the lung toxins paraquat and 4-ipomeanol. By contrast, orciprenaline, salbutamol, and terbutaline were without effect although some response was recorded with isoprenaline. The filtered gas phase of cigarette smoke and acrolein, one of its components, were inhibitory but protection was afforded by N-acetylcysteine. It is suggested that the inhibitory effects of cigarette smoke may be due to its acrolein content. It is further suggested that the use of lung slices and measurements of 14C-leucine incorporation provide valuable means for monitoring potential pulmonary toxins.

Pulmonary toxicity of thioureas in the rat.

Administration of alpha-naphthylthiourea (ANTU) to rats causes damage to pulmonary endothelial cells and possibly mesothelial lining cells that together may account for the massive pleural effusion characteristic of thiourea toxicity. Using 35S-thiourea as a model compound, the extent of binding of 35S to lung proteins correlated well with the extent of edema, suggesting that the extent of binding of thiourea metabolites is a measure of lung toxicity. ANTU and phenylthiourea (PTU) compete for 35S binding to lung slices, suggesting that these toxins may act in a similar way. Binding of 35S in lung slices from resistant rats is much less than in controls, and resistance cannot be explained by differences in either whole body metabolism or redistribution of thiourea in vivo. Lung glutathione levels (in vitro and in vivo) in normal and resistant rats following thiourea administration were essentially the same. However, at doses of thiourea that cause pleural effusion, there was an increase in total lung glutathione.

Examination of the toxicity of several protein toxins of plant origin using bovine pulmonary endothelial cells.

The bovine pulmonary endothelial (BPE) cell line was examined as a model to study the toxicity of ricin and abrin toxins currently under investigation. The BPE cell line was examined because ricin has been shown to bind to endothelial cells. Cell viability was assessed using several different biochemical parameters including growth (DNA by binding of gentian violet stain), mitochondrial function (succinate dehydrogenase activity) using MTT and lysosomal integrity (neutral red retention assay). In order to compare toxicities and investigate potential protective compounds, concentrations of toxins causing death of 50% and 70% of the (control) cell population (LC50 and LC70, respectively) were determined. It is concluded that while ricin and abrin share a common mechanism of action ricin is slightly less toxic than abrin. BPE cells are a good model for future mechanistic studies and particularly for initial phase screening of potentially therapeutic compounds. Carbohydrates were used in an attempt to examine which receptor types were involved in the binding and uptake of ricin and abrin by the cell line. It was found that only high concentrations of galactose prevented lethality while mannose apparently had no effect. Furthermore, the molar excess of carbohydrate to toxin required in order to achieve protection indicated that this would be an impractical approach to adopt in vivo.

The in vitro penetration and distribution of T-2 toxin through human skin.

The penetration and distribution of T-2 toxin in excised human abdominal skin has been determined for a dose range of 1.0-2.6 micrograms/cm2 skin using an ethanol vehicle and a saline receptor solution. In all cases the overall percentage penetration of T-2 after 48 h was low, the greatest amounts of toxin being present in the stratum corneum with less in the epidermis and relatively little in the dermis. Vehicle: skin partition coefficients support this finding. Neither penetration nor distribution were changed by a rabbit serum receptor solution. Electron micrographs showed that at 1.8 micrograms/cm2 and above the contents of the intercellular space are leached out to leave the integument as a porous membrane. The distribution of T-2 within the skin after 48 h would suggest that for doses up to 2.6 micrograms/cm2 the irritative and inflammatory effects on the skin would be of more immediate concern than would systemic toxicity.

Examination of toxicity of Clostridium perfringens -toxin in the MDCK cell line.

The epithelial Madin Darby canine kidney (MDCK) cell line was examined as a model to study the toxicity of Clostridium perfringens -toxin. The MDCK cell line was used because it is a monolayer cell line sensitive to -toxin. Using the neutral red (NR) retention assay (an indicator of lysosomal integrity), the concentration of toxin causing death in 50% of the cell population (LC(50)) was 900 pM, although this was found to vary between production batches. -Toxin was found to act rapidly but with a lag phase of 1 hr (NR assay). Pulsing the cultures with toxin (up to 4800 pM) indicated that the duration of exposure required to exert an effect was potentially very short (2.5 min). Increasing the duration of exposure beyond 3 hr did not decrease cell viability any further. Experiments with protease inhibitors failed to inactivate the toxin. Ethylenediaminetetraacetic acid (EDTA) was found to potentiate the lethality of the toxin by 90% This may be due to non-specific chaotropic effects such as membrane destabilization. By exposing cultures of MDCK cells to -toxin for a second time, resistance to the effects of the toxin was increased by 43%. The factor(s) controlling resistance to the toxin may have a heritable component.

Adenosine accumulation as an indicator of cell-specific toxicity in rat lung slices.

We have investigated the potential of adenosine uptake as a marker of chemically induced, cell-selective pulmonary injury using cell-selective toxicants. The administration of alpha-naphthylthiourea (ANTU), an agent which is known to damage the pulmonary endothelium, diminished spermidine and adenosine accumulation. In contrast, paraquat (a toxicant that selectively damages pulmonary epithelial cells) did not reduce adenosine uptake, although uptake of spermidine (a marker of pulmonary epithelial damage) was reduced. Taken together, these findings suggest that adenosine and spermidine are accumulated into different cell types. We characterized adenosine uptake and fate in rat lung slices. Accumulation was time and concentration dependent. In our experiments, the radiolabel retained within the slice comprised mainly nucleotides, primarily ATP. Adenosine-induced ATP elevation was initially a rapid event which reached a maximum. The use of a well characterized enzymatic assay for ATP confirmed the ATP elevation suggested by thin-layer chromatography (TLC). Adenosine uptake proved a more consistent marker of ANTU-induced pulmonary injury than measurement of 5-hydroxytryptamine (5-HT) uptake, which was slightly increased by ANTU administration to rats compared with control animals.

The absorption of aliphatic carbamates from the rat colon.

The colonic absorptio n of a series of aliphatic carbamates has been studied using an in situ preparation in the rat. The absorption process appeared to be passive and no metabolism by the colon membrane was detected. A linear correlation between the logarithms of the absorption rate constants and apparent partition coefficients was found. The absorption rates were not affected by concurrent water flux.

The relationship between chemical structure and the in vivo metabolism of an homologous series of n-alkyl carbamates.

The metabolism and pharmacokinetics of carbonyl [14C] labelled ethyl, n-butyl, n-hexyl and n-octyl carbamates has been examined in rats after oral and intravenous administration. Hydrolysis of the carbamate group was a major metabolic fate, particularly of the more water soluble carbamates. Conversely, with increasing lipophilicity increasing amounts of omega-1 oxidation products were found both in plasma and urine. The plasma pharmacokinetic data could not be explained by a simple bi-exponential model, ethyl carbamate in particular showing unexpectedly persistent blood levels. A model has been proposed to explain the pharmacokinetic data for ethyl, n-butyl, n-hexyl and n-octyl carbamates. The essential features of this model are that carbamate is thought not to be in equilibrium between the peripheral and central compartment and that hydrolytic metabolism takes place in the peripheral compartment while oxidative metabolism to urinary metabolites occurs in the central compartment.

Gastrointestinal absorption of carbenoxolone in the rat determined in vitro and in situ: deviations from the pH-partition hypothesis.

The absorption of [14C] carbenoxolone from everted rat ileum in vitro and from rat stomach and ileum in situ has been examined. The rate of its mucosal to serosal transfer in vitro increases as pH increases from 5 to 8 whereas the amount bound to ileum tissue decreases with increased pH; absorption closely parallels the drug's solubility. The uptake of carbenoxolone in situ is bi-exponential and the rate constants for the two processes, have been calculated. Absorption in situ, and biliary excretion, of the drug increases with increasing pH from 5.0 to 7.4. Tissue binding to the ileum in situ is not dependent on pH except below pH 5.0 when extensive tissue accumulation of carbenoxolone occurs because of its low solubility. Tissue binding to the stomach increases markedly with decrease of pH from 7.4 to 6.5 and at pH 6.5 is 80 times greater than binding to the intestine. The rate of absorption from the stomach, at pH 6.5-7.4, was much less than that from the intestine in situ. When allowance is made for the binding of carbenoxolone to the stomach, contrary to the pH-partition hypothesis, correlation is apparent between its absorption and the amount present in the ionized form.

The generation of a human dermal equivalent to assess the potential contribution of human dermal fibroblasts to the sulphur mustard-induced vesication response.

1. A human dermal equivalent (HDE) gel was constructed from rat tail tendon collagen (type 1) and human dermal fibroblasts (HFs). Histological studies revealed that the HFs within the HDE gel matrix assumed the shape of differentiated dermal fibroblasts and were metabolically viable as determined by the MTT assay. 2. The HDE system was developed to determine if viable, differentiated HFs have the potential to contribute to tissue damage by releasing the proteolytic enzyme elastase following exposure to sulphur mustard (HD). Elastase was measured, using the substrate suc-ala-ala-val-p-nitroanilide (SAAVNA), because of its association with various human pathological bullous skin diseases. An additional elastase substrate (suc-ala-ala-ala-p-nitroanilide; SAAANA) was also used. A miniaturised assay was employed to measure lactate dehydrogenase (LDH), a cytosolic enzyme released following damage to the cell membrane. 3. Elastase levels (measured with SAAVNA) increased to over 740% of those in control culture medium at 24 h after exposure of the HDE to HD (2 mM) and may therefore be part of the mechanism associated with dermo-epidermal separation and blistering in humans following exposure of skin to HD. LDH was released from the HDE after exposure to HD in a time dependent fashion, suggesting a steady leakage of cytosolic constituents after the initial exposure. 4. The results suggest that differentiated human dermal fibroblasts have the potential to contribute to the development of the vesication response by releasing proteases such as elastase extracellularly after HD exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteine esters protect cultured rodent lung slices from sulphur mustard.

1. In previous studies an in vitro rat lung slice system was used to investigate the metabolic and structural changes after exposure to known lung toxicants. 2. In this study, the same system was used to identify the ability of cysteine esters to protect against sulphur mustard toxicity. 3. The cyclopentyl (CCPE), cyclohexyl (CCHE), isopropyl (CIPE), methyl (CME) esters of cysteine, cystine dimethyl ester (CDME), cysteine (CySH) and N-acetyl cysteine (NAc) were all non-toxic to cultured rat lung slices at 5 mM (equivalent cysteine concentration) after a pretreatment time of 30 min. 4. Pretreatment with the isopropyl, cyclohexyl, cyclopentyl and methyl esters of cysteine at concentrations higher than 1 mM protected against an IC50 of sulphur mustard, however, neither cysteine nor N-acetylcysteine protected. 5. We propose that the extent of protection is directly related to increased levels of intracellular cysteine provided by the esters of cysteine.