Transfer of uremic solutes across the human term placenta: An ex vivo study in the dual-side perfused cotyledon.

INTRODUCTION: An increasing number of women becomes pregnant while suffering from chronic kidney disease (CKD). As a result of decreased renal function, uremic solutes circulate at high levels in the maternal circulation. This study aimed to acquire more knowledge about the placental transfer of uremic solutes across the human placenta. METHODS: Placental transfer was studied in healthy term placentas, via the ex vivo dual-side human cotyledon perfusion technique (closed-closed set-up for both maternal and fetal circulations). Uremic solute concentrations in maternal and fetal perfusates were measured via LC-MS/MS over 180 min of perfusion. RESULTS: We found that the studied compounds demonstrated different degrees of placental transfer. Fetal-to-maternal perfusate ratios at t = 180 min were for anthranilic acid 1.00 ± 0.02, indole-3-acetic acid 0.47 ± 0.08, hippuric acid 0.36 ± 0.18, l-arabinitol 0.33 ± 0.04, indoxyl sulfate 0.33 ± 0.11, neopterin 0.28 ± 0.14 and kynurenic acid 0.13 ± 0.03. All uremic solutes studied also emerged in the perfusates when cotyledons were perfused in the absence of uremic solute concentrations added to the maternal reservoir. For kynurenin these concentrations were so high, it complicated the calculation of a transfer ratio for the exogenously administered compound. DISCUSSION: After 180 min of exposure the extent of placental transfer differs substantially for the solutes studied, reflecting different transfer rates. Future studies should investigate to what extent specific uremic solutes reach the fetal circulation in vivo and how they may interfere with organ function and development of the unborn child.

Placental disposition of the immunosuppressive drug tacrolimus in renal transplant recipients and in ex vivo perfused placental tissue.

Currently, tacrolimus is the most potent immunosuppressive agent for renal transplant recipients and is commonly prescribed during pregnancy. As data on placental exposure and transfer are limited, we studied tacrolimus placental handling in samples obtained from renal transplant recipients. We found transfer to venous umbilical cord blood, but particularly noted a strong placental accumulation. In patient samples, tissue concentrations in a range of 55-82 ng/g were found. More detailed ex vivo dual-side perfusions of term placentas from healthy women revealed a tissue-to-maternal perfusate concentration ratio of 113 ±â€¯49 (mean ±â€¯SEM), underlining the placental accumulation found in vivo. During the 3 h ex vivo perfusion interval no placental transfer to the fetal circulation was observed. In addition, we found a non-homogeneous distribution of tacrolimus across the perfused cotyledons. In conclusion, we observed extensive accumulation of tacrolimus in placental tissue. This warrants further studies into potential effects on placental function and immune cells of the placenta.

Bioengineered kidney tubules efficiently excrete uremic toxins.

The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.

The effect of dipyridamole on the pharmacokinetics of metformin: a randomized crossover study in healthy volunteers.

PURPOSE: Concomitant treatment with the glucose-lowering drug metformin and the platelet aggregation inhibitor dipyridamole often occurs in patients with type 2 diabetes mellitus who have suffered a cerebrovascular event. The gastrointestinal uptake of metformin is mediated by the human equilibrative nucleoside transporter 4 (ENT4), which is inhibited by dipyridamole in preclinical studies. We hypothesized that dipyridamole lowers the plasma exposure to metformin. METHODS: Eighteen healthy volunteers (mean age 23 years; 9 male) were randomized in an open-label crossover study. Subjects were allocated to treatment with metformin 500 mg twice daily in combination with dipyridamole slow-release 200 mg twice daily or to metformin alone for 4 days. After a washout period of 10 days, the volunteers were crossed over to the alternative treatment arm. Blood samples were collected during a 10-h period after intake of the last metformin dose. The primary endpoint was the area under the plasma concentration-time curve (AUC0-12h) and the maximum plasma metformin concentration (C max). RESULTS: In healthy subjects, dipyridamole did not significantly affect Cmax nor AUC0-12h of metformin under steady-state conditions. CONCLUSIONS: Previous in vitro studies report that dipyridamole inhibits the ENT4 transporter that mediates gastrointestinal uptake of metformin. In contrast, co-administration of dipyridamole at therapeutic dosages to healthy volunteers does not have a clinically relevant effect on metformin plasma steady-state exposure. This observation is reassuring for patients who are treated with this combination of drugs.

Ticagrelor Does Not Inhibit Adenosine Transport at Relevant Concentrations: A Randomized Cross-Over Study in Healthy Subjects In Vivo.

BACKGROUND AND PURPOSE: In patients with myocardial infarction, ticagrelor reduces cardiovascular and sepsis-related mortality, and can cause dyspnea. It is suggested that this is caused by adenosine receptor stimulation, because in preclinical studies, ticagrelor blocks the nucleoside transporter and increases cellular ATP release. We now investigated the effects of ticagrelor on the adenosine system in humans in vivo. EXPERIMENTAL APPROACH: In a double-blinded, placebo-controlled cross-over trial in 14 healthy subjects, we have tested whether ticagrelor (180 mg) affects adenosine- and dipyridamole-induced forearm vasodilation, as surrogates of nucleoside uptake inhibition and adenosine formation, respectively. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was measured. Primary endpoint was adenosine-induced vasodilation. KEY RESULTS: Ticagrelor did not affect adenosine- or dipyridamole-induced forearm vasodilation. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was not affected by ticagrelor. In vitro, ticagrelor dose-dependently inhibited nucleoside uptake, but only at supra-physiological concentrations. CONCLUSION AND IMPLICATIONS: In conclusion, at relevant plasma concentration, ticagrelor does not affect adenosine transport, nor adenosine formation in healthy subjects. Therefore, it is unlikely that this mechanism is a relevant pleiotropic effect of ticagrelor. TRIAL REGISTRATION: ClinicalTrials.gov NCT01996735.

Uremic toxins inhibit renal metabolic capacity through interference with glucuronidation and mitochondrial respiration.

During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2µM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13µM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.

Dipyridamole enhances ischaemia-induced reactive hyperaemia by increased adenosine receptor stimulation.

BACKGROUND AND PURPOSE: Dipyridamole enhances post-occlusive reactive hyperaemia (PORH) in the human forearm vascular bed. We hypothesize that this effect is completely mediated by increased adenosine receptor stimulation. To test this hypothesis, the effect of caffeine (an adenosine receptor antagonist) on dipyridamole-induced augmentation of PORH was explored. EXPERIMENTAL APPROACH: The forearm vasodilator responses to three increasing periods of forearm ischaemia (2, 5 and 13 min) were determined during placebo infusion. Forty minutes after the last reperfusion period, this procedure was repeated during intra-arterial infusion of dipyridamole (7.4 nmol min(-1) per 100 ml forearm). At least 2 weeks later, this whole procedure was repeated, but now in the presence of caffeine (90 microg min(-1) per 100 ml volume). KEY RESULTS: After 2, 5 and 13 min of ischaemia, the average forearm blood flow increased to 5.6+/-0.7, 9.7+/-1.3 and 34.5+/-2.1 ml min(-1) per 100 ml. After infusion of dipyridamole into the brachial artery, these numbers were significantly increased to 7.7+/-0.8, 12.5+/-1.5 and 41.6+/-3.1 ml min(-1) per 100 ml. This response was abolished by the concomitant infusion of caffeine (6.6+/-0.5, 10.2+/-0.6, 35.1+/-2.2 (caffeine) versus 7.4+/-0.4, 10.5+/-0.6, 33.7+/-2.2 ml min(-1)per 100 ml (caffeine/dipyridamole)). CONCLUSIONS AND IMPLICATIONS: Caffeine prevented the augmenting effect of dipyridamole on PORH. This indicates that dipyridamole-induced augmentation of PORH is mediated via increased adenosine receptor stimulation as a result of elevated extracellular formation of adenosine during ischaemia.

Methotrexate modulates the kinetics of adenosine in humans in vivo.

BACKGROUND: Animal studies suggest that the anti-inflammatory effect of methotrexate (MTX) is mediated by increased adenosine concentrations. OBJECTIVE: To assess the effect of MTX on the vasodilator effects of adenosine and the nucleoside uptake inhibitor, dipyridamole, in humans in vivo as a marker for changes in adenosine kinetics. METHODS: Ten patients with active arthritis were treated with MTX (15 mg/week). Measurements were performed before and after 12 weeks of treatment. At these time points, the activity of adenosine deaminase was measured in isolated lymphocytes, and forearm blood flow (FBF) was determined by venous occlusion plethysmography during administration of adenosine and dipyridamole into the brachial artery. RESULTS: The Vmax of adenosine deaminase in lymphocytes was reduced by MTX treatment (p<0.05). MTX significantly enhanced vasodilator response to adenosine (0.5 and 1.5 microg/min/dl of forearm tissue; mean (SE) FBF ratio increased from 1.2 (0.2) to 1.4 (0.2) and 2.2 (0.2) ml/dl/min, respectively, before and from 1.3 (0.1) to 1.8 (0.2) and 3.2 (0.5) ml/dl/min during MTX treatment; p<0.05). Also, dipyridamole-induced vasodilatation (30 and 100 microg/min/dl) was enhanced by MTX (FBF ratio increased from 1.2 (0.2) to 1.5 (0.3) and 1.8 (0.2), respectively, before and from 1.3 (0.1) to 1.8 (0.2) and 2.4 (0.4) during MTX treatment; p<0.05). CONCLUSIONS: MTX treatment inhibits deamination of adenosine and potentiates adenosine-induced vasodilatation. Also dipyridamole-induced vasodilatation is enhanced by MTX treatment, suggesting an increased extracellular formation of adenosine. These effects on the adenosine kinetics in humans may contribute to the therapeutic efficacy of MTX.

Role of multidrug resistance protein 2 (MRP2) in glutathione-bimane efflux from Caco-2 and rat renal proximal tubule cells.

1. The multidrug resistance protein 2 (MRP2) has been shown to play an important role in the transport of glutathione conjugates in the liver. Its importance in renal excretion, however, is still uncertain and other organic anion transporters may be involved. The objective of the present study was to characterize glutathione conjugate efflux from rat kidney proximal tubule cells (PTC), and to determine the contribution of Mrp2. 2. We used isolated PTC in suspension, as well as grown to monolayer density. For comparison, transport characteristics were also determined in the human intestinal epithelial cell line Caco-2, an established model to study MRP2-mediated transport. The cells were loaded with monochlorobimane (MCB) at 10 degrees C. MCB enters the cells by simple diffusion and is conjugated with glutathione to form the fluorescent glutathione-bimane (GS-B). 3. In primary cultures of rat PTC, no indications for a transporter-mediated mechanism were found. The efflux of GS-B from Caco-2 cells and freshly isolated PTC was time- and temperature-dependent. Furthermore, GS-B transport in both models was inhibited by chlorodinitrobenzene (CDNB), with an inhibitory constant of 46.8+/-0.9 microM in freshly isolated PTC. In Caco-2 cells, the inhibitory potency of CDNB was approximately 20 fold higher. Finally, efflux of GS-B from freshly isolated PTC from Mrp2-deficient (TR(-)) rats was studied. As compared to normal rat PTC, transport characteristics were not different. 4. We conclude that in freshly isolated rat PTC glutathione conjugate excretion is mediated by other organic anion transporters rather than by Mrp2.

Biological fate of [14C]-1-nitropyrene in rats following intragastric administration.

1-Nitropyrene (1-NP), a weak carcinogen associated with diesel exhaust particles, has previously been detected in workplace atmospheres with in-use diesel engines and in the general environment. In order to gain insight in its biological fate, a single dose of [14C]-1-NP (27.6 microCi, 750 mg/kg body weight, b.w.) was administered intragastrically to rats and the presence of metabolites in blood and tissue homogenates, and radioactivity associated with blood proteins and tissue DNA, were studied. Early peak levels of radioactivity observed in blood and tissue homogenates indicated a rapid absorption of [14C]-1-NP from the gastrointestinal tract. Metabolite patterns observed in plasma, liver and kidney homogenates strongly suggested an important role of the intestinal microflora in the enterohepatic recirculation, but not in nitroreduction of 1-NP prior to absorption from the gastrointestinal tract. This might explain the low levels of radioactivity associated with blood proteins, since 1-nitrosopyrene, a product of nitroreduction of 1-NP, is likely to be involved in protein binding. Levels of radioactivity associated with plasma proteins were approximately four times higher than the levels of radioactivity associated with hemoglobin (401.0 and 84.1 pmol/g protein per micromol 1-NP kg b.w., respectively, at 24 h). Maximal 25% of the associated radioactivity was released following mild alkaline hydrolysis of either hemoglobin or plasma proteins. 1-Aminopyrene was the only released compound after hydrolysis of hemoglobin. In addition to 1-aminopyrene, two more polar unidentified metabolites were detected following hydrolysis of plasma proteins. Association of radioactivity with DNA was highest in the liver at the first moments of observation (7.4 pmol 14C Eq./mg DNA per micromol 1-NP kg b.w.), but decreased rapidly to levels lower than observed for kidney DNA (max. 3.0 pmol 14C Eq./mg DNA per micromol 1-NP kg b.w. at 24 h). In lungs 8-50 times less radioactivity was associated with DNA than observed in the liver and kidneys. The results of this study show, that 1-NP undergoes an extensive and complex biotransformation in vivo, resulting in a variety of metabolites present in blood and tissue homogenates and a diversity of blood protein adducts. Concentrations of plasma metabolites, blood protein adducts and DNA adducts were rather low. In addition, previous studies also showed relatively low concentrations of metabolites present in urine. Therefore, sensitive and selective methods will be needed in order to evaluate the biological fate of 1-NP, associated with diesel exhaust particles, in humans.

Sensitive and selective detection of urinary 1-nitropyrene metabolites following administration of a single intragastric dose of diesel exhaust particles (SRM 2975) to rats.

1-Nitropyrene (1-NP) has been proposed as a marker for exposure to diesel exhaust particles (DEP). Since the extent of the actual intake of 1-NP adsorbed on DEP will be relatively low, sensitive and selective methods are needed regarding human exposure assessment. Two analytical methods are presented for the assessment of 1-NP metabolites in urine of male Sprague-Dawley rats administered a single intragastric dose of native DEP (SRM 2975, 20 mg, 35.7 microgram of 1-NP/g). Enzymatically hydrolyzed urine was extracted using Blue Rayon. The extracts were analyzed directly, using HPLC with postcolumn on-line reduction and fluorescence detection (HPLC-Flu), or were processed further for GC/MS/MS analysis. Although sensitive to several metabolites, the HPLC-Flu method lacked selectivity for quantitation of some important metabolites in rat urinary extracts, and therefore seems suitable for screening purposes only. With regard to GC/MS/MS analysis, derivatization with heptafluorobutyrylimidazole (HFBI) yielded low limits of determination for hydroxy-1-aminopyrenes, hydroxy-N-acetyl-1-aminopyrenes (converted to derivatized hydroxy-1-aminopyrenes by the reagent), and 1-aminopyrene (1.8-9.2 fmol on the column). Derivatization of hydroxy-1-nitropyrenes yielded relatively high limits of determination, and therefore, hydroxy-1-nitropyrenes were reduced to hydroxy-1-aminopyrenes prior to derivatization with HFBI. Intragastric administration of DEP to rats resulted in urinary excretion of 6-hydroxy-N-acetyl-1-aminopyrene, 8-hydroxy-N-acetyl-1-aminopyrene, 6-hydroxy-1-nitropyrene, 8-hydroxy-1-nitropyrene, and 3-hydroxy-1-nitropyrene (7, 1.2, 1.6, 0.3, and 0.5% of the dose within 12 h, respectively). 1-Nitropyrene, N-acetyl-1-aminopyrene, and 3-, 6-, and 8-hydroxy-1-aminopyrene were not observed as urinary metabolites following administration of a single dose of DEP. The observed excretion pattern and urinary metabolite concentrations suggest that 1-NP present on unmodified DEP becomes bioavailable to a large extent and is metabolized in the same way as was previously observed following administration of pure 1-NP. The presented methods are promising for assessment of human exposure to 1-NP, e.g., following exposure to DEP, because of the possibility of analyzing large volumes of urine, the conversion of three types of metabolites to one (the amino metabolites), and the low detection limits that are achieved.

Determination of hemoglobin adducts following oral administration of 1-nitropyrene to rats using gas chromatography-tandem mass spectrometry.

1-Nitropyrene (1-NP) has successfully been used as a marker for environmental monitoring of exposure to diesel exhaust. This study presents a sensitive and selective method for detection of Hb adducts after oral administration of a single dose 1-NP to rats, by measuring 1-aminopyrene (1-AP) after in vitro hydrolysis of the adducts. Released 1-AP was extracted with hexane and derivatized with heptafluorobutyric acid anhydride prior to GC-MS-MS analysis. Optimal conditions for the release of 1-AP were hydrolysis under nitrogen, in 1 M NaOH at 70 degrees C for 60 min. Analysis of a stock solution of Hb adducts of 1-NP utilizing these conditions showed to be reproducible over a period of several weeks with a coefficient of variance of 9.5%. The determination limit was 10-20 pg 1-AP per 70-90 mg globin. A study of the time course of Hb adduct formation showed a fast absorption and an early peak concentration of released 1-AP, approximately 39 pg 1-AP/mg globin at 3 h after exposure. After the maximum was reached, 1-AP concentrations decreased bi-phasically. Initially a fast decline was observed, followed by a slow decrease to 5.9+/-1.9 pg 1-AP/mg globin at 24 h after administration.

Influence of Aroclor 1254, phenobarbital, beta-naphthoflavone, and ethanol pretreatment on the biotransformation of cyclophosphamide in male and female rats.

The aim of the present study is to investigate the influence of the environmental factors, smoking and alcohol, on the biotransformation of cyclophosphamide (CP) in the rat in vivo and in vitro with S9 liver fractions. The biotransformation of CP was studied by the determination of the CP metabolites, nor-nitrogen mustard (NNM), 4-ketocyclophosphamide (KCP), and carboxyphosphamide (CAR). The effect of the environmental factors, smoking and alcohol consumption, on the biotransformation enzymes was mimicked by pretreatment of rats with beta-naphthoflavone and ethanol, respectively. Rats treated with olive oil and water served as controls and rats pretreated with Aroclor 1254 and phenobarbital were used as positive controls. The influence of sex and supplementation with NAD and GSH, mimicking a biological variation in NAD and GSH levels in rat and human liver, was also studied. Pretreatment of rats with Aroclor 1254 decreased the excretion of unmetabolized CP in urine, most likely due to an enhanced biotransformation. The in vitro hepatic biotransformation of CP in rats was strongly influenced by sex, by supplementation with NAD and GSH, and by pretreatment with the enzyme-inducers, phenobarbital and Aroclor 1254. No influence of pretreatment with the enzyme-inducers, beta-naphthoflavone and ethanol, was found. The results suggest that the influence of the environmental factors, alcohol consumption and smoking, on the biotransformation of CP in man will be negligible.

Determination of cyclophosphamide metabolites by gas chromatography and thermionic specific detection. Interindividual differences in hepatic biotransformation of cyclophosphamide in man in vitro.

Sensitive methods for the determination of the cyclophosphamide metabolites nornitrogen mustard, 4-ketocyclophosphamide and carboxyphosphamide are presented. After liquid-liquid extraction and derivatization, the metabolites are determined by gas chromatography and thermionic specific detection. The methods were used to study the in vitro biotransformation of cyclophosphamide with S-9 liver fractions of human donors. The results show large interindividual differences in the formation of nornitrogen mustard and carboxyphosphamide. 4-Ketocyclophosphamide was not detected.

Effects of the peroxisome proliferator mono(2-ethylhexyl)phthalate in primary hepatocyte cultures derived from rat, guinea pig, rabbit and monkey. Relationship between interspecies differences in biotransformation and peroxisome proliferating potencies.

Primary hepatocyte cultures derived from rat, rabbit, guinea pig and monkey have been treated in vitro with metabolites of di(2-ethylhexyl)phthalate, i.e. mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (metabolite V) and mono(2-ethyl-5-oxohexyl)phthalate (metabolite VI). In rat hepatocyte cultures MEHP and metabolite VI were equally potent in inducing peroxisome proliferation, while metabolite V was much less potent. In rat hepatocytes a 50% increase in both peroxisomal palmitoyl-CoA oxidase activity and microsomal lauric acid omega-hydroxylation activity was found after treatment with 5-15 microM MEHP. In guinea pig, rabbit and monkey hepatocyte cultures, a 50% increase in peroxisomal palmitoyl-CoA oxidase activity was found after treatment with 408-485 microM MEHP. No induction of lauric acid omega-hydroxylation activity was found. These results indicate that peroxisome proliferation can be induced by MEHP in rabbit, guinea pig and monkey hepatocytes, but that these species are at least 30-fold less sensitive to peroxisome proliferation induction than rats. The proposed mechanistic inter-relationship between induction of lauric acid omega-hydroxylation activity and peroxisome proliferation is found in rat hepatocytes, but not in hepatocytes of the other three species. Treatment of guinea pig hepatocyte cultures with MEHP resulted in an increase in triglyceride concentrations in the hepatocytes. In rat and rabbit hepatocyte cultures, triglyceride concentrations were much less altered by MEHP. In monkey hepatocytes a decrease in hepatic triglyceride concentration was found after treatment with MEHP. These effects are in agreement with in vivo effects observed before. After treatment of primary hepatocyte cultures with MEHP, high concentrations of omega- and (omega-1)-hydroxylated metabolites of MEHP were found in media from rat, rabbit and guinea pig cultures. The formation of these metabolites did not decline in time. During treatment the metabolite profile in media from rat hepatocyte cultures moved towards omega-hydroxy metabolites of MEHP. In media from monkey hepatocyte cultures the lowest concentrations of hydroxylated metabolites were determined. No major species differences were found in the potency to form oxidized MEHP metabolites, and thus no unique metabolite differences were found, which could explain the species differences in sensitivity for peroxisome proliferation.

Metabolites of the plasticizer di(2-ethylhexyl)phthalate in urine samples of workers in polyvinylchloride processing industries.

Little is known about occupational exposure to the plasticizer di(2-ethylhexyl)phthalate (CAS number 117-81-7), a compound widely used in polyvinylchloride (PVC) plastics. We have studied the uptake of DEHP in workers by determining the concentrations of four metabolites of DEHP in urine samples, i.e., mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)phthalate. In addition DEHP concentrations in the air were determined by personal air sampling. Nine workers in a PVC boot factory exposed to a maximum of 1.2 mg/m3 DEHP showed an increase in the urinary concentrations of all four metabolites over the workshift. These results were obtained on both the first and the last day of the workweek. With the exception of MEHP, the increases in the concentrations of the metabolites during a workday were statistically significant. Six workers from a PVC cable factory exposed to a maximum of 1.2 mg/m3 DEHP showed a one- to fourfold increase in the concentrations of the four metabolites over the workshift, but these increases were not statistically significant. These results indicate that measurement of DEHP metabolites in urine samples may be of use for monitoring the occupational exposure to DEHP.

Determination of four metabolites of the plasticizer di(2-ethylhexyl)phthalate in human urine samples.

A method for biological monitoring of exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) is described. In this method the four main metabolites of DEHP [i.e., mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)-phthalate] are determined in urine samples. The procedure includes enzymatic hydrolysis, ether extraction, and derivatization with triethyloxonium tetrafluoroborate. Analysis is performed by gas chromatography-electron impact mass spectrometry. The detection limit for all four metabolites is less than 25 micrograms/l urine. The coefficient of variation based on duplicate determinations of urine samples of workers occupationally exposed to DEHP was 16% for MEHP (mean concentration 0.157 mg/l) and 6%-9% for the other three metabolites (mean concentrations 0.130-0.175 mg/l). The method described here was used to study DEHP metabolism in man. Most persons excrete mono(2-ethyl-5-oxohexyl)-phthalate and mono(2-ethyl-5-hydroxyhexyl)phthalate as a (glucuronide) conjugate. Mono(5-carboxy-2-ethylpentyl)phthalate is mainly excreted in free form, while for MEHP a large interindividual variation in conjugation status was observed. Of the four metabolites quantified, 52% are products of a (omega-1)-hydroxylation reaction of MEHP [i.e., mono(2-ethyl-5-oxohexyl)phthalate and mono(2-ethyl-5-hydroxylation reaction of MEHP [i.e., mono(5-carboxy-2-ethylpentyl)phthalate], and 26% is not oxidized further (i.e., MEHP). A good correlation is obtained when the amount of MEHP omega-hydroxylation products is compared with the amount of MEHP (omega-1)-hydroxylation products in urine samples. When the internal dose of DEHP has to be established we recommend that the levels of all four metabolites of DEHP be studied in urine samples.

Microsomal lauric acid hydroxylase activities after treatment of rats with three classical cytochrome P450 inducers and peroxisome proliferating compounds.

In order to investigate a proposed relationship between induction of hepatic microsomal lauric acid hydroxylase activity and peroxisome proliferation in the liver, male Wistar rats were treated with peroxisome proliferating compounds, and the lauric acid hydroxylase activity, the immunochemical detectable levels of cytochrome P450 4A1 and the activities of peroxisomal enzymes were determined. In addition, the levels of cytochrome P450 4A1 and lauric acid hydroxylase activities were studied after treatment of rats with three cytochrome P450 inducers. After treatment with aroclor-1254, phenobarbital or 3-methylcholanthrene total cytochrome P450 was 1.7-2.7 times induced. However, no induction of lauric acid omega-hydroxylase activities or P450 4A1 levels were found. After treatment of rats with di(2-ethylhexyl)phthalate (DEHP) a dose-dependent induction of lauric acid omega-hydroxylase activities, levels of cytochrome P450 4A1 and peroxisomal fatty acid beta-oxidation was found. Even at a dose-level of 100 mg DEPH/kg body weight per day a significant induction of these activities was observed. The main metabolites of DEHP, mono(2-ethylhexyl)phthalate and 2-ethyl-1-hexanol, also caused an induction of levels of P450 4A1, lauric acid omega-hydroxylase activities and the activity of peroxisomal palmitoyl-CoA oxidase. 2-Ethyl-1-hexanoic acid did not influence lauric acid omega-hydroxylase activities, but did induce levels of P450 4A1 and palmitoyl-CoA oxidase activities. Three other compounds (perfluoro-octanoic acid, valproate and nafenopin) induced both lauric acid omega-hydroxylase activity and peroxisomal palmitoyl-CoA oxidase activity. The plasticizer, di(2-ethylhexyl)adipate, did not induce levels of P450 4A1, lauric acid omega-hydroxylase activities or palmitoyl-CoA oxidase activities. With the compounds tested a close association between the induction of lauric acid omega-hydroxylase activities and peroxisomal palmitoyl-CoA oxidase activity was found. These data support the theory that peroxisome proliferating compounds do induce lauric acid omega-hydroxylase activities and that there might be a mechanistic inter-relationship between peroxisome proliferation and induction of lauric acid omega-hydroxylase activities.

Detection of contamination with antineoplastic agents in a hospital pharmacy department.

The contamination with fluorouracil, cyclophosphamide and methotrexate was studied in a hospital pharmacy department where these drugs were prepared. In the preparation room, air samples were taken before and during preparation of the drugs. Methotrexate was detected in one sample which was collected during preparation (0.3 micrograms/m3). Spot samples were taken in the vertical laminar airflow safety hood before and after preparation of the drugs and after cleaning of the hood. Contamination of the laminar airflow hood was: cyclophosphamide: 1-160 ng/cm2; fluorouracil: 10-62 ng/cm2 and methotrexate: 2-633 ng/cm2. Spot samples from the floor in front of and beneath the laminar airflow hood showed contamination with especially fluorouracil (48-236 micrograms/m2). The gloves used during preparation of the drugs were contaminated mainly with fluorouracil (5-980 ng/cm2). Urine samples from two workers involved in the preparation of the drugs were analysed for unmetabolized cyclophosphamide; it was not detected. Although no uptake of cyclophosphamide was established, it is shown that the methods for measurement of cyclophosphamide, fluorouracil and methotrexate in the preparation room are applicable for the control of occupational exposure to these drugs.

Urinary cyclophosphamide excretion in rats after intratracheal, dermal, oral and intravenous administration of cyclophosphamide.

The urinary excretion of unmetabolized cyclophosphamide was studied in rats after intratracheal, dermal, oral and intravenous administration. Rats were given two doses of 1 mg kg-1 cyclophosphamide 48 h apart and urine was collected for 96 h after the first treatment. With the help of a phosphor-specific filter in a flame photometer attached to a gas chromatograph, low levels of cyclophosphamide were determined after derivatization with trifluoroacetic anhydride. Cumulative excretion as a percentage of dose ranged from 4.0 to 6.9 after the first dose and 2.7 to 5.5 after the second dose. The highest rate of excretion after the second administration was observed in rats treated intratracheally, while cumulative excretion was higher (6.9%) after the first than after the second (2.7%) intravenous treatment. The most prolonged excretion was observed after dermal application.