4',5'-unsaturated 5'-halogenated nucleosides. Mechanism-based and competitive inhibitors of S-adenosyl-L-homocysteine hydrolase.

The design and synthesis of (E)- and (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (6 and 13, respectively), a new class of mechanism-based inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase, is described. A number of analogues of 6 and 13 were synthesized in order to determine the structure-activity relationship necessary for inhibition of the enzyme. Substitution of chlorine for fluorine in 6 (i.e. 44), addition of an extra chlorine to the 5'-vinyl position (i.e. 51 and 52), modification of the 2'-hydroxyl group to the deoxy (34 and 35) and arabino (36 and 37) nucleosides provided competitive inhibitors of SAH hydrolase. Nucleosides 6 and 13, as well as 5'-deoxy-5',5'-difluoroadenosine (14) proved to be time-dependent inhibitors of SAH hydrolase. All three compounds are postulated to inhibit through the potent electrophile derived from oxidation of the 3'-hydroxyl of 6 or 13 to the ketone (i.e. 3 and/or the E-isomer). Consistent with the proposed mechanism of inactivation of SAH hydrolase by 6, 13, and 14 was the observation that incubation of purified rat liver SAH hydrolase with 6 resulted in release of 1 equiv of fluoride ion (by 19F NMR) and incubation with 14 resulted in release of 2 equiv of fluoride ion. The general synthetic route developed for the synthesis of the title nucleosides utilized the fluoro Pummerer reaction for the introduction of fluorine into the requisite precursors. Preliminary antiretroviral data from Moloney leukemia virus (MoLV) is presented and correlates with SAH hydrolase inhibition. Antiviral activity (IC50 against MoLV) ranged from 0.05 to 10 micrograms/mL.

2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase.

It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.

Antimalarial activity of a 4',5'-unsaturated 5'-fluoroadenosine mechanism-based inhibitor of S-adenosyl-L-homocysteine hydrolase.

A 4',5'-unsaturated 5'-fluoroadenosine inhibitor of S-adenosyl-L-homocysteine hydrolase (SAH hydrolase; EC 3.3.1.1), MDL 28842, was found to inhibit markedly the growth of Plasmodium falciparum in vitro and Plasmodium berghei in mice. Inhibition of P. berghei growth was associated with a large increase in the concentration of S-adenosyl-L-homocysteine (SAH) in the erythrocytes of the mice treated with MDL 28842. This increase in SAH was due apparently to inhibition of the mouse erythrocyte SAH hydrolase activity, because SAH hydrolase activity was undetectable in either P. berghei or P. falciparum isolated from infected erythrocytes, although enzyme activity was readily detected in mouse erythrocyte extracts. Therefore, MDL 28842 probably inhibits plasmodial growth indirectly by adversely changing the milieu of the host erythrocyte. SAH hydrolase represents a worthwhile target for the future development of potent inhibitors for the chemotherapy of malaria.

Depletion of estrogen receptor in human breast tumor cells by a novel substituted indole that does not bind to the hormone binding domain.

Steroidal antiestrogens appear to have at least two major modes of action in breast cancer cells, direct antagonism of estrogen binding to its receptor and depletion of estrogen receptors (ER) due to inhibition of dimerization of the receptor and a resultant destabilization of the receptor protein. In a search for other classes of compounds which would act as dimerization inhibitors, a novel substituted indole (8-{2-[1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-1H-indol-3-yl]-acetylamino} octanoic acid butyl-methyl amide, MDL 101,906) was synthesized. Binding of the ER to its consensus response element (ERE) was apparently decreased in nuclear extracts from MCF-7 human breast cancer cell treated with MDL 101,906. This decreased binding was found to be due to depletion of ER based on direct measurement of ER using an enzyme-linked immunoassay. Other transcription factors were apparently unaffected by MDL 101,906 treatment. Whereas depletion of ER with a steroidal antiestrogen was almost complete after 3 h of treatment of MCF-7 cells, the effect of MDL 101,906 took significantly longer to occur, suggesting a fundamental difference in the mechanisms of action of the two drugs. This was also evident in the lack of binding of MDL 101,906 to the hormone binding domain of ER. MDL 101,906 treatment also caused depletion of ER mRNA in MCF-7 cells. Depletion of ER mRNA was noted by 3 h of drug treatment and was apparently almost complete after 24 h of treatment. Depletion of ER from MCF-7 cells led to a dose-dependent decrease in the expression of luciferase by an ERE-driven luciferase reporter gene assay system. The mechanism of MDL 101,906 appears to be unique and additional studies with this chemical class seem to be warranted to assess the potential for therapeutic utility.

Effect of route of nutrition on recovery of hepatic organic anion clearance after fasting.

BACKGROUND: Previous work documented a 40% depression of hepatic indocyanine green (ICG) clearance (ClICG) in pigs fasted to 20% weight loss, with return to normal within 12 days of food refeeding. ClICG in pigs is insensitive to changes in hepatic blood flow but very sensitive to changes in hepatic function (HF). Serial ClICG determinations were performed to quantify the effect of route of nutrient delivery on recovery of HF. METHODS: Fourteen pigs were fasted to 20% weight loss (12.8 days average) with both gastrostomy and intravenous catheters placed in each animal midway through the fast. ClICG was measured before fast, after fast, and after 12 days refeeding through the enteral or parenteral route at 125 kcal/kg/day with isonitrogenous, isocaloric diets containing 9% fat. Urine and stool were analyzed for total nitrogen. RESULTS: No significant differences appeared between groups in nitrogen output during fasting (4.5 +/- 1.2 gm/kg enteral, 4.6 +/- 1.2 gm/kg parenteral), in nitrogen intake (800 +/- 19 mg/kg/day enteral, 810 +/- 10 mg/kg/day parenteral), or in before or after fast ClICG, but enteral feeding produced more positive nitrogen balance. ClICG improved significantly with enteral but not with parenteral feeding. CONCLUSIONS: Enteral feeding produces faster nitrogen accrual and reverses the depression of major pathways of bilirubin and organic anion excretion associated with malnutrition. Parenteral feeding failed to improve organic anion clearance despite weight gain.

Antiretroviral activity of mechanism-based irreversible inhibitors of S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine hydrolase (AdoHcy-nase) is a key enzyme in transmethylation reactions. The objective of the present study was to examine the potential antiretroviral activities of novel mechanism-based irreversible AdoHcy-nase inhibitors. (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (ZDDFA), (E)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (EDDFA), (Z)-4',5'-didehydro-5'-deoxy-5'-chloroadenosine (ZDDCA) and 5'-deoxy-5'-acetylenic adenosine (DAA) inhibited AdoHcy-nase activity with Ki values of 0.55, 1.04, greater than 10.0 and 3.30 microM, respectively. These four compounds were tested for antiviral activity in vitro against Moloney leukemia virus (MoLV) in the XC-plaque assay. MoLV replication in murine fibroblasts (SC-1) was inhibited by ZDDFA, EDDFA and DAA with IC50 values of 0.05, 0.25 and 3.30 micrograms/ml, respectively. ZDDCA did not inhibit MoLV infection at the concentrations tested. Antiviral activity correlated with the ability of the individual compounds to maintain sustained elevations in intracellular S-adenosylhomocysteine (AdoHcy) concentrations in the SC-1 cells. ZDDFA, the most potent inhibitor of AdoHcy-nase and MoLV was also the most active in maintaining sustained elevations in intracellular AdoHcy levels. The antiviral activity of ZDDFA was also examined in murine C3H1OT1/2 fibroblasts which constitutively produce MoLV. Pretreatment with ZDDFA (1.0 microgram/ml) for 24 hr inhibited virus production by 88%. Similar to the SC-1 cells, and concomitant with enzyme inhibition, there was a 300-fold increase in AdoHcy levels in ZDDFA (1.0 microgram/ml) treated C3H1OT1/2 cells. Incorporation of a [3H]methyl group from tritiated S-adenosylmethionine into total RNA in C3H1OT1/2 cells was inhibited by ZDDFA without affecting cell viability. These results suggest that mechanism-based inhibitors of AdoHcy-nase, such as ZDDFA, may have potential as antiretroviral agents.

Piezoelectric quartz crystal biosensors.

Biosensing methods utilize the intrinsic selectivity of a biorecognition process to create relatively simple, low cost, analytical alternatives for a variety of research investigations. Here, biosensor applications of the piezoelectric quartz crystal (PQC) are reviewed. The discussion is divided into sections focusing on the development of PQC based analytical techniques, applications in solution phase sensing pertaining to PQC biosensors, and the current state of knowledge in PQC biosensing applications. Immobilization procedures, dip and dry assay techniques, and solution phase sensing methods are considered in detail.

Atropine and ephedrine adsorption to syringe plastic.

The purpose of this study was to compare daily changes in the concentration of atropine or ephedrine sulfate solutions that had been stored up to 4 days in plastic or glass syringes. Sets of three plastic and one glass syringe were used for each drug; the glass syringes acted as controls. Each set of syringes was labeled as day 0, 1, 2, 3, or 4. Syringes with medication were laid horizontally, had needles attached, and were stored in the dark at an ambient temperature. Each day, the assigned set of syringes was analyzed by high-performance liquid chromatography. Results showed that the change in the ephedrine sulfate concentration in the plastic syringes from day 0 to day 4 was less than 1.4%. Atropine sulfate decreased 52% over 4 days, with the largest single drop occurring during the first 24 hours. It can be concluded that the two brands of ephedrine sulfate stored up to 4 days at ambient temperature in the brand of syringe used do not significantly decrease in concentration. However, this was not the case with the brand of atropine sulfate studied. The practice of storing atropine sulfate in plastic syringes should be discouraged, because of the possibility of loss of potency due to medication adsorption to syringe plastic.

DNA-protein cross-link formation in Burkitt lymphoma cells cultured with benzaldehyde and the sedative paraldehyde.

Exposure to aldehydes represents potential risks to human and animal health. Cyclic aldehydes such as benzaldehyde, 2-furaldehyde, and paraldehyde were found to induce formation of stable DNA-protein cross-links (DPXs) in cultured human lymphoma cells. A relationship between increased cytotoxicity and DPX formation was observed with each aldehyde. Paraldehyde is a sedative drug used predominately in treatment of ethanol withdrawal. Paraldehyde was the most potent cross-linking aldehyde studied, yet least cytotoxic. Although DPX formation by aliphatic aldehydes is well-known, this study confirms the potential for cyclic aldehydes to cause formation of DPXs in cultured cells at therapeutically relevant doses.

Effect on dissolution from halving methylphenidate extended-release tablets.

OBJECTIVE: To determine the effect on in vitro dissolution from cutting methylphenidate extended-release tablets in half. DESIGN: Ritalin-SR (Ciba Pharmaceutical Co.) and generic methylphenidate extended-release (MD Pharmaceutical Inc.) tablets were dissolved in water according to the method prescribed by the US Pharmacopeia under two conditions: whole and halved. Samples were collected at 15, 30, and 45 minutes and at 1, 2, 3, 3.5, 4, 5, 6, and 7 hours. Methylphenidate content was determined by HPLC. RESULTS: Halving the tablets caused a statistically significant increase in cumulative dissolution as early as 15 minutes. The difference in cumulative dissolution reached its maximum for both Ritalin-SR and generic methylphenidate extended-release tablets at 2 hours. At this time point, the percent dissolution of the whole versus halved tablets was 57% versus 74% (Ritalin-SR), respectively, and 49% versus 67% (generic), respectively. The dissolution profiles of halved and whole extended-release methylphenidate tablets were parallel from this point through the 7-hour collection period. At 7 hours, however, there was no difference in the cumulative dissolution of halved versus whole tablets. CONCLUSIONS: While statistical differences during in vitro dissolution do exist and pharmacokinetic ramifications have not yet been determined, the absolute differences in dissolution between halved and whole tablets are not great. Halving methylphenidate extended-release tablets may be a clinically acceptable means of achieving a small increment/decrement in dose without converting to a regular-release tablet.

Simultaneous quantification of ethylmorphine O-deethylase and N-demethylase activity by high-performance liquid chromatography.

The N-demethylation and O-deethylation of ethylmorphine by cytochrome P-450 are simultaneously measured using high-performance liquid chromatography. All the metabolites and the substrate are extracted from the enzymatic incubation mixture with isopropanol-methylene chloride (20:80) containing 6.0 micrograms/ml codeine sulfate as an internal standard. Separation of the compounds is achieved on a C18 reversed-phase column using a mobile phase of 1% acetic acid--acetonitrile (85:15) with 1-hexanesulfonic acid as a counter-ion. Total run time is 12 min at a flow-rate of 2.0 ml/min and 144 bar. Assay of ethylmorphine N-demethylase and O-deethylase activities in rat liver microsomes revealed close agreement between this method and conventional ones. N-Demethylation was found to greatly exceed O-deethylation in liver microsomes from either control or phenobarbital-treated rats confirming results from other laboratories. This method can also be used to measure the N- and O-demethylation of codeine.

A piezoelectric method for monitoring formaldehyde induced crosslink formation between poly-lysine and poly-deoxyguanosine.

To date, no experimental technique has been used to monitor DNA-protein crosslink formation in real-time. Real-time data is important for understanding the underlying chemical mechanisms associated with this reaction process. Here, the novel adaptation of existing piezoelectric quartz crystal (PQC) or quartz crystal microbalance (QCM) technology was used to monitor, in real-time, the formation of a crosslink bond induced by formaldehyde between lysine and guanine. Previous results showed complexes of lysine and guanine constitute a major portion of the DNA-protein crosslinks formed. Thus, poly-lysine(5) and poly-deoxyguanosine(11) were used as a model system to develop this detection method. Poly-lysine(5) was immobilized on QCM electrode surfaces by covalent attachment through polyethylenimine (PEI). Immobilization was confirmed by the decrease in dry QCM frequency; data consistency suggested uniform coatings were produced. The QCM sensor was configured within a thermostatic environmental chamber. The system was calibrated and baseline responses to variations in the analyte solution matrix were identified. QCMs with immobilized poly-lysine(5) were placed in contact with formaldehyde and poly-deoxyguanosine(11), and crosslink formation was monitored in real-time. Crosslink formation was verified through evaluation of controls. Control assays indicated some of the frequency signal was as aresult of non-specific association. Further assays were conducted after saturation of non-specific binding. This real-time data represents a significant advancement in the state of knowledge of the crosslinking process and provides the experimental foundation for further QCM crosslink investigations.

Relative potency of mexiletine, lidocaine, and tocainide as inhibitors of rat liver CYP1A1 activity.

Mexiletine and tocainide are lidocaine congeners that share similar chemical structures. Clinical studies suggest that the in vivo inhibitory effect of mexiletine on the CYP1A family of isoforms is substantially greater than that of tocainide. We investigated the inhibitory property of mexiletine, lidocaine, and tocainide on the in vitro activity of the cytochrome P4501A1 (CYP1A1) isozyme in the rat. Hepatic microsomes were prepared from rat livers induced with 3-methylcholanthrene. The rate of ethoxyresorufin-O-dealkylation (EROD) was used as an index of CYP1A1 activity. Vmax and KM of the reactions were determined from Lineweaver-Burk plots. The Ki values for the inhibitors were derived from Dixon plots. Results showed that mexiletine is a competitive inhibitor, lidocaine is a mixed inhibitor, and tocainide is a noncompetitive inhibitor of EROD. The Ki values for mexiletine and tocainide were 0.30 +/- 0.02 mM and 12.4 +/- 0.7 mM, respectively. Two Ki values for lidocaine were determined. They were 0.65 +/- 0.07 mM and 4.1 +/- 1.3 mM, respectively. The relative inhibitory potency of these agents on rat CYP1A1 activity is mexiletine > lidocaine > tocainide. This difference in potency, which is most likely attributable to the change in the chemical composition in the aliphatic chain among the compounds, suggests that these compounds may be useful probes for studying the mechanism of the interaction with the active site of CYP1A1.

Brain mitochondrial citrate synthase and glutamate dehydrogenase: differential inhibition by fatty acyl coenzyme A derivatives.

Organic acidemia is found in several metabolic encephalopathies (e.g., hepatic and valproate encephalopathies, Reye's syndrome, and hereditary organic acidemias). Although fatty acids are known to be neurotoxic, the underlying mechanisms have not been fully elucidated. It has been hypothesized that one mechanism underlying fatty acid neurotoxicity is the selective inhibition of rate-limiting and/or regulated tricarboxylic acid (TCA) cycle and related enzymes by fatty acyl-coenzyme A (CoA) derivatives. To test the hypothesis, this study has examined the effects of several fatty acyl-CoAs on citrate synthase (CS) and glutamate dehydrogenase (GDH) in brain mitochondria. At levels higher than 100 microM, butyryl-CoA (BCoA; a short-chain acyl-CoA; IC50 approximately 640 microM), octanoyl-CoA (OCoA; a medium-chain acyl-CoA; IC50 approximately 380 microM), n-decanoyl-CoA (DCoA; a medium-chain acyl-CoA; IC50 approximately 436 microM), and palmitoyl-CoA (PCoA; a long-chain acyl-CoA; IC50 approximately 340 microM) inhibited brain mitochondrial CS activity in a concentration-related manner. However, these fatty acyl-CoAs were less effective inhibitors (IC50 values for OCoA, DCoA, and PCoA being approximately 1260, 420, and 720 microM, respectively) of brain mitochondrial GDH activity. Compared to the other three acyl-CoAs investigated, BCoA was a very poor inhibitor of GDH. These results demonstrate that fatty acyl-CoAs are inhibitors of brain mitochondrial CS and GDH activities only at pathological/toxicological levels. Thus, the fatty acyl-CoA inhibition of brain mitochondrial CS and GDH is unlikely to assume major pathophysiological and/or pathogenetic importance.(ABSTRACT TRUNCATED AT 250 WORDS)

Gas chromatographic-mass spectrometric analysis of plasma nifedipine.

A gas chromatographic-mass spectrometric assay is described for the determination of plasma nifedipine using nitrendipine as an internal standard. Chromatographic separation was performed on a dimethylsilicone capillary column, and detection was by selected ion monitoring of electron impact-generated base peak ions. The lower limit of quantifiable detection of nifedipine was 2 ng/ml. The method was applied to plasma samples obtained from a human subject who had been dosed with a 10-mg nifedipine capsule every 8 h for eight doses.

The mechanism of inhibition of S-adenosyl-L-homocysteine hydrolase by fluorine-containing adenosine analogs.

(Z)-4',5'-Didehydro-5'-deoxy-5'-fluoroadenosine (I), 5'-deoxy-5'-difluoroadenosine (II), and 4',5'-didehydro-5'-deoxy-5'-fluoroarabinosyl-adenosine (III) are inhibitors of rat liver S-adenosyl-L-homocysteine hydrolase. Compounds I and II are time-dependent and irreversible inhibitors of the enzyme. Both I and II are oxidized by E.NAD to produce E.NADH, and fluoride anion is formed in the inactivation reaction (0.7 to 1.0 mole fluoride/mole of enzyme subunit, and 1.7 moles fluoride/mole of enzyme subunit from I and II, respectively). The enzyme is stoichiometrically labeled with [8-3H]-I, but the label is lost upon denaturation of the protein either with or without treatment of the labeled complex with sodium borohydride. The compound III, the arabino derivative of I, is a competitive inhibitor of the enzyme. The mechanism of the inhibition of S-adenosyl-L-homocysteine hydrolase by these inhibitors is discussed.

Differential effects of fatty acyl coenzyme A derivatives on citrate synthase and glutamate dehydrogenase.

We investigated the hypothesis that one mechanism underlying fatty acid toxicity is the selective inhibition of rate-limiting and/or regulated tricarboxylic acid cycle and related enzymes by fatty acyl coenzyme A (CoA) derivatives by examining the effects of several fatty acyl CoAs on purified citrate synthase (CS) and glutamate dehydrogenase (GDH). The results indicate that, at pathophysiological levels, palmitoyl CoA, a long-chain acyl CoA, is a potent inhibitor of CS and GDH with IC50 values of 3-15 microM. At much higher levels (in the pathological and toxicological range), octanoyl and decanoyl CoA (medium-chain acyl CoAs) inhibited both enzymes with IC50 values of 0.4-1.6 mM. Butyryl CoA, a short-chain acyl CoA, inhibited CS (IC50 = 0.9 mM) at toxicological levels but inhibited GDH poorly. These results suggest that the long-chain fatty acyl CoA inhibition of CS and GDH may assume some pathophysiological importance in fatty acid toxicity and in metabolic encephalopathies in which organic acidemia is persistent. The findings also provide additional support for the original hypothesis.

Bioequivalence of immediate-release theophylline capsules.

A three-way crossover study in 18 healthy male volunteers was conducted to evaluate the bioequivalence of three different 200 mg anhydrous theophylline immediate-release (IR) capsules. The products had not been rated as therapeutically equivalent by the US Food and Drug Administration (FDA) owing to a lack of bioequivalence data. Serum samples were obtained from 0 to 34 h after dosing. Mean time of maximum serum concentration (T(max)) ranged from 1.3 to 1.4 h. Mean values for the maximum serum concentration (C(max)) and the area under the serum concentration-time curves (AUC) differed by <5% for the three products. The confidence limits for Ln-transformed C(max) and AUC ranged from >/=89 to

Selective inhibition of T cell activation by an inhibitor of S-adenosyl-L-homocysteine hydrolase.

Defects in the enzymes involved in the pathway of S-adenosylmethionine (AdoMet) metabolism, or inhibition of those enzymes, results in profound immunodeficiency. We have examined MDL 28,842, a novel irreversible inhibitor of S-adenosyl-L-homocysteine hydrolase (AdoHcyase), an enzyme involved in AdoMet metabolism, to determine its effect on the immune system and to investigate its potential as an immunosuppressive agent. The stimulation of human mononuclear cell proliferation in vitro with Con A, a T cell mitogen, and PWM, a T-dependent B cell mitogen, were inhibited by MDL 28,842. The 50% inhibitory concentration for both were 0.33 microM. In murine spleen cells, MDL 28,842 was a potent, nontoxic, inhibitor of Con A-stimulated T cell proliferation (IC50 = 0.19 microM) but did not affect LPS-induced B cell proliferation. This selective suppression was also observed when enriched murine T and B cells were stimulated with mitogens, although S-adenosyl-L-homocysteine (AdoHcy), the substrate of AdoHcyase, was similarly elevated in both populations. In addition to proliferation in response to a number of stimuli, IL-2 production and the expression of IL-2R by mitogen-stimulated T cells were inhibited by MDL 28,842. These results suggest a direct effect of MDL 28,842 on T cells. In vivo, the antibody response to a T cell-dependent Ag, OVA, was inhibited by MDL 28,842. The response of splenic T cells from these animals to OVA in vitro were similarly depressed compared with controls. The results demonstrate that MDL 28,842 is a potent nontoxic immunosuppressive agent, which has selectivity for T cells and therefore may be useful in the treatment of T cell-mediated disorders, such as autoimmune disease and tissue transplantation.

Measurement of DNA-protein cross-links in human leukocytes following acute ingestion of chromium in drinking water.

Increased DNA-protein cross-linking (DPX) in circulating leukocytes has been proposed as a potential biomarker for exposure and genotoxic damage caused by inhalation of certain reactive chemicals, such as hexavalent chromium [Cr(VI)]. This study was designed to determine whether ingestion of a single dose of potassium dichromate alone [Cr(VI)] or potassium dichromate fully reduced to Cr(III) with orange juice (prior to ingestion) causes an increase in DPX of circulating leukocytes in humans. Four adult male volunteers ingested a bolus dose of 5000 micro chromium in a 0.51 volume of water (10 p.p.m.), and blood samples were collected at 0, 60, 120, 180 and 240 min afterwards for analysis of DPX formation in circulating leukocytes. Results were compared to each person's own background concentration of DPX in leukocytes. Blood and urine samples were also collected for up to 2 weeks following the dose to examine the pattern of uptake and excretion of chromium. The results showed that there was no significant change in DPX observed following either Cr(VI) or Cr(III) ingestion, even though blood and urine chromium measurements indicated systemic uptake of a substantial fraction of the ingested chromium. Since Cr(III) does not possess DPX-inducing properties while Cr(VI) does, these results suggest that the Cr(VI) was reduced to Cr(III) intragastrically prior to absorption or that the amount of Cr(VI) absorbed into the blood was insufficient to produce DPX. These results are consistent with prior research that indicated that DPX would not occur following exposure to Cr(VI) except at very high doses.