Drug protein conjugates—I. A study of the covalent binding of [14C]captopril to plasma proteins in the rat

The metabolism of [¹⁴C]captopril has been investigated in vitro and in vivo in male Wistar rats. The formation of conjugates of [¹⁴C]captopril with plasma proteins was observed both in vitro and in vivo: 180 min after intravenous infusion of [¹⁴C]captopril 35 ± 5% of total radioactivity was covalently bound to plasma proteins. The fate of [¹⁴C]captopril-plasma protein conjugates was investigated in vivo. [¹⁴C]Captopril was incubated in vitro with rat and human plasma and the resulting captopril-protein conjugates were infused into male rats. The plasma concentration of [¹⁴C]captopril-rat plasma protein conjugates declined monoexponentially with a half-life of 71.1 ± 2.2 min. After 180 min 28 ± 3% of the radioactivity was excreted in urine, largely as [¹⁴C]captopril-cysteine mixed disulphide (67%). Thus although captopril readily forms covalent bonds with plasma proteins the resulting conjugates dissociate in vivo. The toxicological implications of these findings are discussed.     

Metabolism and excretion of 2,4-[14C]dinitrotoluene in conventional and axenic Fischer-344 rats

Comparisons of in vitro reduction of 2,4-dinitrotoluene (2,4-DNT) by cecal microflora and liver have indicated that microflora may play a large role in the in vivo metabolism of 2,4-DNT to reduced metabolites. Furthermore, reduction of 2,4-DNT by cecal microflora produces nitroso and, presumably, hydroxylamino intermediates which may account for the toxic actions of 2,4-DNT, including hepatocarcinogenesis. This study examines the metabolism, excretion, and hepatic covalent binding of 2,4-DNT in conventional, DNT-fed, and axenic Fischer-344 rats in order to define more precisely the role of DNT pretreatment and intestinal microflora in the disposition and toxicity of 2,4-DNT. No differences in 2,4-DNT disposition were produced by 30 days of feeding DNT (35 mg/kg/day) in the diet of male or female rats. Axenic males and females excreted less of a dose of 2,4-DNT in the urine than did conventional animals, and half-times for excretion of 4-(N-acetyl)amino-2-nitrobenzoic acid (4NAC2NBA), 2,4-dinitrobenzoic acid, 2-amino-4-nitrobenzoic acid (2A4NBA), and 2,4-dinitrobenzyl alcohol glucuronide were longer in axenic males than in conventional males. In axenic females half-times for excretion of only 4NAC2NBA and 2A4NBA were longer than in conventional females. Amounts of 4NAC2NBA and 2A4NBA excreted by axenic animals were 1/10th to 1/5th those excreted by conventional animals. Hepatic covalent binding was decreased by half in axenic animals. These data suggest that intestinal microflora play a major role in the appearance of reduced urinary metabolites and of covalently bound material after 2,4-DNT administration.     

Induction of hepatic microsomal drug-metabolizing enzymes by sulfur-containing metabolites of chlorinated benzenes in rats

3,5-Dichlorophenyl methyl sulfide and its oxidized compounds, metabolites of m-dichlorobenzene, increased the activity of aniline hydroxylase and aminopyrine N-demethylase and the content of cytochromes P-450 and b₅ in rat liver microsomes. Hexobarbital sleeping time was reduced by these three compounds. After the administration of the sulfide to rats, 3,5-dichlorophenyl methyl sulfone appeared in blood, liver, and kidneys, and remained detectable in the blood and the tissues between 120 and 240 hr. The sulfone was considered to play a principal role in the induction by these compounds. Other chlorophenyl methyl sulfones also showed similar induction activity.     

High-affinity binding of [3H]doxepin to histamine H1-receptors in rat brain: Possible identification of a subclass of histamine H1-receptors

The binding of the radioactively labeled tricyclic antidepressant, [³H]doxepin, to rat brain tissie was examined. Scatchard plots of specific [³H]doxepin binding indicated the presence of two distinct binding sites. The equilibrium dissociation constant (K_D) of the high-affinity site was 0.020 nM with a maximal binding capacity (B_max) of 13.7 fmol/mg protein. The corresponding values for the low-affinity site were 3.6 nM and 740 fmol/mg protein, respectively. The high-affinity site was sensitive to competition by pharmacologically relevant concentrations of histamine H₁ antagonists such as pyrilamine (K_D = 1.0 nM), diphenhydramine (K_D = 20 nM), d-chlorpheniramine (K_D = 1.7 nM), and 1-chlorpheniramine (K_D = 97 nM). The B_max for [³H]doxepin binding in the high-affinity H₁-receptor, however, was approximately 10% of the B_max obtained using [³H]pyrilamine to label the H₁-receptor. Various tricyclic antidepressants were very potent inhibitors at the high-affinity [³H]doxepin site. Their potencies, however, did not correlated with their potencies previously reported for the H₁-receptor. The regional distribution of [³H]doxepin high-affinity sites correlated with the known distribution of H₁-receptors in the rat brain. These results suggest that [³H]doxepin is binding to a subclass of histamine H₁-receptors.     

Metabolism, disposition and excretion of [C]melamine in male Fischer 344 rats

The metabolism, excretion and disposition of melamine were determined after administration of a single oral dose of 0.025 mCi (0.38 mg) [¹⁴C]melamine to adult male Fischer 344 rats. Within the first 24 hr, 90% of the administered dose was excreted in the urine. Negligible radioactivity appeared in breath and faeces. There was little difference in blood, liver or plasma concentrations of ¹⁴C, suggesting that melamine distributes in body water. The only organs showing radioactivity levels much higher than plasma were the kidney and bladder. The bladder level was by far the highest, a finding probably due either to back diffusion from urine or to contamination of bladder tissue with urine. Virtually no residual radioactivity was observed in tissues examined at 24 hr or later. The elimination-phase half-life calculated from plasma data, 2.7 hr, was in good agreement with the urinary-excretion half-life of 3.0 hr. The renal clearance of melamine was 2.5 ml/min. Radioactivity in plasma or urine co-chromatographed with that of the dosing solution, indicating that melamine is not metabolized in the male Fischer 344 rat.     

Influence of strain differences in mice on the metabolism and toxicity of benzene

Chronic benzene exposure results in a progressive depression of bone marrow function and is thought to be caused by a metabolite of benzene (Snyder and Kocsis, 1975; Goldstein and Laskin, 1977). Several reports concerning differences in xenobiotic metabolism and toxicity among inbred strains of mice prompted us to study benzene metabolism and toxicity in C57BL/6 and DBA/2 mice. DBA/2 mice were more susceptible to benzene than C57BL/6 mice. No differences in the total amount of urinary benzene metabolites produced were found between the strains; however, differences in the relative amounts of specific metabolites were noted. DBA/2 mice produced more phenylglucuronide but less ethereal sulfate conjugates than C57BL/6 mice. Hydrolysis of the urinary conjugates revealed that DBA/2 mice excreted more phenol, but less hydroquinone than C57BL/6 mice. Multiple dose studies revealed that the more resistant C57BL/6 mice contained less water soluble benzene metabolites in bone marrow, liver, kidney, blood, spleen, and lung than DBA/2 mice. C57BL/6 mice also contained less covalently bound metabolites in bone marrow, blood, spleen, and muscle than DBA/2 mice following multiple doses of benzene. V_max values for UDPGA utilization in C57BL/6 mice were almost six times the V_max values observed for DBA/2 mice. Furthermore, V_max values for phenylsulfate formation in C57BL/6 mice were three times the V_max values for DBA/2 mice. It was concluded that the difference in susceptibility to benzene between C57BL/6 and DBA/2 mice was not the result of a single factor, buth rather, the sum total of a number of metabolic events.     

Standardization and calibration of whole-body autoradiography for routine semiquantitative analysis of the distribution of 14C-labeled compounds in animal tissues

A procedure is described and evaluated for the standardization and densitometric calibration of whole-body autoradiography (WBAR) using ¹⁴C-labeled compounds. Calibration was achieved using uniform ¹⁴C-labeled poly(methyl)methacrylate standards. The mathematical relationship between activity and autoradiographic image density was linear, resulting in a convenient method for the comparison of relative radioactivity in tissues. Temperature and duration of autoradiographic exposure effected the slope but not the linearity of this relationship. The range of linearity of this function allows for a comparison of radioactivity over two orders of magnitude. The effects of section thickness on autoradiographic density were evaluated and determined to be linear over a range of from 10 to 100 μm. It was concluded that this procedure provides a reliable and reproducible semiquantitative method for the routine evaluation of compound or metabolite distribution and can be a valuable adjunct to pharmacokinetic studies.     

Effects of pectin-containing diets on the hepatic macromolecular covalent binding of 2,6-dinitro-[3H]toluene in Fischer-344 rats

The influence of diets varying in pectin content on intestinal microfloral metabolic capacity of rats has been investigated as a possible mechanism for the alteration of toxicity of 2,6-dinitrotoluene (2,6-DNT) produced by these diets. Male F-344 rats were fed a purified diet (AIN-76A), AIN-76A plus 5% or 10% citrus pectin, or either of two cereal-based diets that vary in pectin content, NIH-07 or Purina Chow 5002. After 28 days, rats were given tritium-labeled 2,6-DNT (10 or 75 mg/kg po) and killed 12 hr later. Total hepatic macromolecular covalent binding (CVB) was determined by exhaustive extraction. The CVB of 2,6-DNT was found to be independent of diet at 10 mg/kg. However, at 75 mg/kg CVB was increased 40% by feeding 5% pectin in the purified diet and 90% by feeding 10% pectin in the purified diet. Animals fed Purina 5002 and NIH-07 had 135 and 150% higher CVB, respectively, than animals fed the purified diet alone and significantly greater CVB than animals fed the pectin supplemented diets. Elevated (two- to threefold) β-glucuronidase and nitroreductase activities, microfloral enzymes proposed to be involved in the activation of 2,6-DNT to a toxicant, were found in the cecal contents of animals fed the pectincontaining diets which correlated with a two- to threefold increase in total number of cecal anaerobes. These results suggest that pectin-induced changes in microflora may enhance hepatoxicity after high doses of 2,6-DNT.     

The metabolism by rainbow trout (Salmo gairdnerii) of p,p'-[14C]DDT on some of its possible degradation products labeled with 14C

Groups of 5 or 10 Rainbow trout fry (Salmo gairdnerii) weighing around 1 g were injected im with ¹⁴C-labeled 2,2,bis(p-chlorophenyl)-1,1,1-trichloroethane (p,p′-DDT); 2,2,bis(p-chlorophenyl)-1,1-dichloroethylene (p,p′-DDE); 2,2,bis-(p-chlorophenyl)-1,1-dichloroethane (p,p′-DDD); 2,2,bis(p-chlorophenyl)-1-chloroethane (p,p′-DDMS); 1,1-bis(p-chlorophenyl)ethylene (p,p′-DDNU); 2,2,bis(p-chlorophenyl)-1-chloroethylene (p,p′-DDMU); 2,2,-bis(p-chlorophenyl)ethanol (p,p′-DDOH); p-dichlorobenzophenone (p,p′-DBP); p-dichlorobenzhydrol (p,p′-DBH); or bis(p_chlorophenyl) acetic acid (p,p′-DDA). At intervals of 24 hr and 3 weeks after injection (except in the case of the p,p′-[¹⁴C]DDOH- and p,p′-[¹⁴C]DDA-injected groups, where only 24-hr samples were taken), the fish were killed, extracted individually, and analyzed for p,p′-DDT metabolites. Their water was also analyzed 24 hr after injection. p,p′-[¹⁴C]DDT was converted to p,p′-DDE and, to a lesser extent, p,p′-DDD. p,p′-[¹⁴C]DDD was converted to a limited extent to p,p′-DDMU, and p,p′-[¹⁴C]DDMS was similarly dehydrochlorinated to p,p′-DDNU. p,p′-[¹⁴C]DBH and p,p′-[¹⁴C]DBP were interconverted to some extent, and p,p′-[¹⁴C]DDA was converted to p,p′-DBP. p,p′-[¹⁴C]DDOH was apparently converted to p,p′-DBP in one experiment, but a second exposure did not confirm this. None of the dehydrochlorination products (p,p′-[¹⁴C]DDE, p,p′-[¹⁴C]DDMU, and p,p′-[¹⁴C]DDNU) was degraded and p,p′-DDMU therefore appears to be one of the end products of DDT metabolism by trout.     

Complete amino acid sequence of a phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The acidic phospholipase A₂ has been isolated from Naja naja atra (Taiwan cobra) venom. The reduced and S-carboxymethylated enzyme was digested with trypsin, and eleven tryptic peptides which accounted for the whole molecule were isolated by a combination of chromatographic and gel filtration procedures, and their amino acid sequences were determined. The alignment of those eleven tryptic peptides was established by the analysis of nine major peptides obtained by Staphylococcus aureus protease digestion of the reduced and S-carboxymethylated enzyme. The phospholipase A₂ of Naja naja atra is a single polypeptide chain consisting of 120 amino acid residues including 14 intramolecularly linked half-cystines. It shows about 86% (103 out of 120) homology when compared with the Naja mossambica mossambica CM-II enzyme.     

Effect of microsomal enzyme inducers on the soluble enzymes of hepatic phase II biotransformation

Numerous xenobiotics induce microsomal enzymes such as cytochrome P-450-dependent monooxygenases, epoxide hydrolase, and UDP-glucuronyltransferase by causing an increase in enzyme synthesis. Since induction of soluble enzymes involved in phase II biotransformation has not been thoroughly studied, effects of the following microsomal enzyme inducers on three important soluble enzymes were examined: phenobarbital (PB), 3-methylcholanthrene (3-MC), butylated hydroxyanisole (BHA), isosafrole (ISF), pregnenolone-16α-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and trans-stilbene oxide (TSO). Representative microsomal enzymes of phase I pathways were examined simultaneously to indicate effective induction. The inducers selected produced the expected increases in hepatic cytochrome P-450 (75–170%), ethylmorphine (200–260%), and benzphetamine (100–260%) N-demethylation, benzo[a]pyrene hydroxylation (300%), ethoxyresorufin O-deethylation (2700%), and styrene oxide hydration (100–270%). The soluble conjugative enzymes studied were glutathione S-transferase, N-acetyltransferase, and sulfotransferase. Glutathione conjugation of 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, and sulfobromophthalein was increased by TSO (100–160%), BHA (60–80%), ISF (60–80%), and PB (60–80%). β-Naphthylamine N-acetyltransferase activity was increased by PCN and 3-MC (60 and 40%, respectively). Only PCN was able to enhance sulfotransferase. Sulfation of 2-naphthol, taurolithocholate, and dehydroepiandrosterone was increased by 28, 111, and 140%, respectively. In conclusion, while microsomal enzymes could be readily induced, activity of soluble phase II enzymes was increased to a much lesser extent. Of the inducers studied, PCN was the most effective at increasing activity of soluble enzymes.     

Metabolism of 1-[14C]nitropyrene in isolated perfused rat livers

1-Nitropyrene (1-NP), a constituent of diesel exhaust, is carcinogenic to rats and is a bacterial and mammalian mutagen. Biliary and fecal excretion of 1-NP metabolites are the major routes of excretion in rats, suggesting that hepatic metabolism plays a dominant role in determining the biological fate of 1-NP. The purpose of this investigation was to quantitate 1-[14C]NP metabolites formed in isolated perfused rat livers and excreted in bile from rats. Perfused rat livers displayed a capacity for oxidation, reduction, acetylation, and conjugation of 1-NP (or its metabolites). Reduction of 1-NP followed by N-acetylation was the major metabolic pathway observed in the perfused livers. Acetylaminopyrene (AAP) was the major metabolite detected, with total quantities (150 nmol) accounting for about 60% of the total 1-[14C]NP dose (258 nmol) added to the perfusate. Considerably smaller quantities of aminopyrene and hydroxynitropyrenes were also detected. Livers perfused with 1-[14C]NP excreted about 36 nmol equivalents of 1-[14C]NP (12% of the total 1-NP dose) in bile after 60 min. Some of the biliary metabolites were tentatively identified as metabolites of the mercapturic acid pathway. The spectrum of biliary metabolites was qualitatively identical to that seen in bile from intact rats. Quantities of 14C covalently bound to hepatic macromolecules from perfused livers were 0.4 nmol 1-NP eq/g liver. The data from this study indicate that the liver may be an important site for metabolism of 1-NP.     

Binding of adrenergic ligands ([3H]clonidine and [3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [³H]ligands, we studied the binding of [³H]clonidine and [³H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [³H]clonidine (with K_D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [³H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [³H]WB-4101 were also detected in the human frontal cortex (with K_D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [³H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [³H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [³H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [³H]WB-4101 sites was highest in the hippocampus.     

Covalent binding of [14C]methoxychlor metabolite(s) to rat liver microsomal components

[14C]Methoxychlor was incubated with NADPH-fortified liver microsomes from male rats, and covalent binding to microsomal components was determined. The binding process was markedly enhanced when microsomes from phenobarbital-treated rats were employed. However, when microsomes from methylcholanthrene-treated rats were used the level of binding was not significantly affected. Incubation in the presence of glutathione, cysteine, or ascorbate markedly diminished binding. Metyrapone and SKF 525-A, inhibitors of hepatic cytochrome P-450-linked monooxygenase activity, inhibited the binding. Also, ethylmorphine and hexobarbital, alternate substrates of the monooxygenase system, inhibited binding. There was no binding to microsomal components in the absence of NADPH or oxygen. TCPO (1,1,1-trichloropropane-2,3-oxide), an inhibitor of epoxide hydrase activity, failed to enhance the binding process. However, N,N'-diphenyl-p-phenylenediamine (NDP) and n-propyl gallate (PG), both free radical scavengers, decreased binding at micromolar concentrations without altering the extent of formation of polar [14C]methoxychlor metabolites. It was concluded that methoxychlor undergoes a hepatic microsomal monooxygenase(s)-mediated activation and that the resultant reactive metabolites (possibly free radicals) bind covalently to microsomal components. By contrast, the binding resulting from the incubation of an impure mixture of polar [14C]methoxychlor metabolites with liver microsomes did not require NADPH and O2 and was not affected by NDP, Pg, ascorbate, or heat-treatment of microsomes. This finding suggested that the binding subsequent to the initial metabolic activation of methoxychlor does not require further enzymatic transformation. However, whether the binding with metabolites represents the same chemical species as the binding with [14C]methoxychlor remains to be established.     

Changes in some rat hepatic microsomal components induced by prolonged administration of chloroquine

Alterations in microsomal drug metabolizing enzymes and phospholipids following prolonged exposure to chloroquine have been investigated. The levels of microsomal aminopyrine-N-demethylase, aniline hydroxylase and both microsomal and cytosolic glutathione-S-transferase are reduced in treated rats. Microsomal epoxide hydrolase is unaffected by the treatment. An increase in the cholesterol-phospholipid ratio and a decrease in the phosphocholine-sphingomyelin ratio occur. There is a general reduction in the total microsomal phospholipid level though the percentage content of sphingomyelin is higher in all cases. The reduction in microsomal phospholipid level is probably due to a reduced incorporation of acetate into microsomal phospholipids as shown in this study using radioactive sodium acetate.     

Effects of dietary cabbage,brussels sprouts, Illicium verum,Schizandra chinensis and alfalfa on the benzo[a]pyrene metabolic system in mouse liver

Male C57B16 mice were fed on diets containing either 20% cabbage, 20% Brussels sprouts, 20% alfalfa, 5% Schizandra chinensis or 5% Illicium verum (two Chinese medicinal herbs) or on a chow or purified basal diet for 14 days after a 1-wk equilibration period on the basal diet. Liver microsomal fractions were assayed for cytochrome P-450 content, aryl hydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH). Liver microsome-mediated benzo[a]pyrene (BP) metabolism (with and without an EH inhibitor, 1,2-epoxy-3,3,3-trichloropropane) was analysed by HPLC. Liver weights of the animals fed on Brussels sprouts and I. verum were significantly increased compared with those of the animals fed on basal diets. S. chinensis induced a 3-fold increase in cytochrome P-450 (P < 0.05). Although P-450 induction in the other groups was as high as 1.8-fold (for chow), statistical significance was not established. Chow induced AHH activity 2.2-fold (P < 0.05), while S. chinensis and alfalfa induced 1.6-fold and 1.7-fold increases, respectively, in AHH activity, although neither increase was statistically significant. EH was stimulated significantly in the following order: I. verum (2.1-fold) > chow (1.7-fold) >S. chinensis (1.6-fold) > Brussels sprouts (1.4-fold). Total levels of BP metabolism and phenol II (primarily 3-hydroxybenzo[a]pyrene) formation were closely associated for each dietary treatment. Total BP metabolism was significantly increased (2.1-fold) in the chow-fed group and increased 1.6-fold in the S. chinensis group (P > 0.05). No increase was seen with the other diets. Phenol II formation relative to total metabolites was significantly increased for the S. chinensis and I. verum groups compared to the basal group. Diet-related variations in phenol production relative to total metabolism were eliminated by addition of the EH inhibitor to the incubation media.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.     

Development of a radioimmunoassay for [DES-ARG9]-bradykinin

Antisera to [des-Arg9]-bradykinin were elicited in rabbits immunized with the peptide conjugated to thyroglobulin and/or ovalbumin. Sera were screened for the presence of antibody with three radioactive antigens, mono-125I-labeled derivatives of [Tyr1, des-Arg]-kallidin, [Tyr, des-Arg]-bradykinin, and [Tyr, des-Arg]-bradykinin that were prepared by treating mono-125I-labeled [Tyr]-kallidin, [Tyr]-bradykinin, and [Tyr]-bradykinin with carboxypeptidase B. Of the six animals immunized, five produced antibodies to [des-Arg]-bradykinin as evidenced by the ability of their sera to bind at least 33% of the added radioactivity at a final dilution of 1:500. Sensitivity and specificity studies were performed with each labeled antigen and a dilution of antiserum that bound 30-50% of the radioactivity. The best labeled antigen-antibody combination, with respect to titer, sensitivity, and specificity was obtained with [mono-125I-Tyr, des Arg]-bradykinin and serum from a rabbit immunized with [des-Arg]-bradykinin conjugated to ovalbumin with toluene diisocyanate. The lowest concentration of [des-Arg]-bradykinin inhibiting 50% of this radioactive antigen binding was 0.23 ng/ml and the lowest concentration which could be distinguished from no [des-Arg]-bradykinin added was 67 pg/ml. This antiserum cross-reacts with bradykinin and lysyl-bradykinin about 9% but not with methionyl-lysyl-bradykinin.     

Modulation of the immune response to Listeria monocytogenes by benzene inhalation

Benzene is a potent bone marrow toxicant. While all blood cell types are targets for benzene poisoning, lymphocytes are particularly sensitive. The immunotoxic consequences of benzene or its metabolites have been demonstrated in a number of in vitro studies; however, little data exist regarding the effects of benzene on host resistance to infectious agents. This investigation examined the effects of benzene on murine resistance to an infectious agent, Listeria monocytogenes. Four concentrations of benzene were employed, 10, 30, 100, and 300 ppm. To determine recovery from the effects of benzene, two exposure regimens were employed: 5 days prior to infection (preexposure), or 5 days prior to and 7 days during infection (continuous exposure). Appropriate air controls were maintained. Splenic bacterial counts and immune responsive cell populations were determined from mice killed at Days 1, 4, and 7 of infection. Preexposure to benzene produced increased bacterial numbers at Day 4 of the infection only at the highest benzene concentration (300 ppm). In contrast, continuous exposure produced increased bacterial numbers at Day 4 of infection at all but the lowest benzene concentration (10 ppm). Bacteria counts were not increased in any benzene-treated group at Day 1 or Day 7 of infection. The increased bacterial numbers at Day 4 suggest an effect on cell-mediated immune responses. Both T and B lymphocytes were particularly sensitive to benzene exhibiting reductions at all concentrations greater than or equal to 30 ppm for both exposure regimens. Esterase-positive cells, however, were relatively resistant to benzenes effects. The results point to a benzene-induced delay in the immune response to L. monocytogenes.