J. Noordhoek

Environmental contamination and assessment of exposure to antineoplastic agents by determination of cyclophosphamide in urine of exposed pharmacy technicians: is skin absorption an important exposure route?

In the Netherlands, special guidelines and safety precautions were introduced about 10 y ago for preparation and administration of antineoplastic agents. However, little is known about the effectiveness of these measures. In this study, occupational exposure to antineoplastic agents of nine pharmacy technicians who were involved in drug preparation was investigated. Cyclophosphamide, 5-fluorouracil, and methotrexate accounted for 95% of the antineoplastic agents prepared; therefore, the presence of these compounds was monitored. During preparation, cyclophosphamide was detected in the air of the work environment (< 0.04-10.1 micrograms/m3). Contamination of and permeation through latex gloves were found for each of the three compounds. The uptake of cyclophosphamide was assessed by the determination of cyclophosphamide in urine. The drug was found in urine samples of six pharmacy technicians, including three persons who were not directly involved in the preparation of cyclophosphamide. The amounts excreted ranged from 0.2 to 19.4 micrograms/24 h. The results strongly suggest that inhalation is of minor importance for internal exposure, compared with other, presumably dermal, routes.

Mechanisms of drug transfer across the human placenta.

In this review we summarized literature data on the mechanisms of human placental drug transport studied in the isolated perfused placental cotyledon, placental membrane vesicles or trophoblastic cell cultures. Overall human placental drug transport rarely exceeds the transfer of flow‐dependent and membrane‐limited marker compounds. Interestingly, relatively often placental drug transfer appeared to be much smaller, indicating impaired trans-placental transport, depending on the physico-chemical characteristics of the drug or placental factors such as tissue binding or metabolism. Although in perfusion studies overall human placental drug transport occurs by simple diffusion, at the membrane level several drug transport systems have been found, mainly for drugs structurally related to endogenous compounds.

Quinone toxicity in DT-diaphorase-efficient and -deficient colon carcinoma cell lines

The human colon carcinoma cell lines Caco-2 and HT-29 were exposed to three structurally related naphthoquinones. Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM) redoxcycle at similar rates, NQ is a stronger arylator than MEN, and DIM does not arylate thiols. The Caco-2 cell line was particularly vulnerable to NQ and MEN and displayed moderate toxic effects of DIM. The HT-29 cell line was only vulnerable to NQ and MEN after inhibition of DT-diaphorase (DTD) with dicoumarol, whereas dicoumarol did not affect the toxicity of quinones to Caco-2 cells. DTD activity in the HT-29 and Caco-2 cell lines, as estimated by the dicoumarol-sensitive reduction of 2,6-dichlorophenolindophenol, was 393.7 +/- 46.9 and 6.4 +/- 2.2 nmol NADPH x min(-1) x mg protein(-1), respectively. MEN depleted glutathione to a small extent in the HT-29 cell line, but a rapid depletion similar to Caco-2 cells was achieved when dicoumarol was added. The data demonstrated that the DTD-deficient Caco-2 cell line was more vulnerable to arylating or redoxcycling quinones than DTD-expressing cell lines. Exposure of the Caco-2 cell line to quinones produced a rapid rise in protein disulphides and oxidised glutathione. In contrast to NQ and DIM, no intracellular GSSG was observed with MEN. The relatively higher levels of ATP in MEN-exposed cells may account for the efficient extrusion of intracellular GSSG. The reductive potential of the cell as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction was only increased by MEN and not with NQ and DIM. We conclude that arylation is a major contributing factor in the toxicity of quinones. For this reason, NQ was the most toxic quinone, followed by MEN, and the pure redoxcycler DIM elicited modest toxicity in Caco-2 cells.

Gas chromatographic-mass spectrometric determination of nitro polycyclic aromatic hydrocarbons in airborne particulate matter from workplace atmosphere

Abstract A method is described for the determination of 1-nitropyrene and 2-nitrofluorene in low volume ambient air samples (1–150 m3). This method is bas

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.

Prevention of oxidant-induced cell death in Caco-2 colon carcinoma cells after inhibition of poly(ADP-ribose) polymerase and Ca2+ chelation: involvement of a common mechanism.

The human colon carcinoma cell line Caco-2 was exposed to the oxidative stress-inducing agents menadione (MEN), 2,3-dimethoxy-1,4-naphthoquinone, and hydrogen peroxide. All three agents caused DNA damage which was assessed by alkaline unwinding. Further, all three agents induced intensive NAD+ depletion, followed by a decrease in intracellular ATP and viability. Inhibition of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) by 3-aminobenzamide prevented the depletion of NAD+. These cells had a higher viability and ATP content. The most pronounced effect was observed with 25 microM of MEN, while at higher levels a partial preservation of NAD+ was observed with no effect on ATP or viability. The chelation of intracellular calcium by bis-(o-aminophenoxy)-ethane-N,N,N1,N1-tetraacidic acid/tetraacetoxymethyl) ester also prevented the dramatic loss of NAD+, demonstrating that Ca2+ is an activating factor in PARP-mediated cell killing.

Cytotoxicity of menadione and related quinones in freshly isolated rat hepatocytes: effects on thiol homeostasis and energy charge

The cytotoxic events in freshly isolated rat hepatocytes following exposure over 2 h to menadione (2-methyl-1,4-naphthoquinone) and two closely related quinones, 2,3-dimethyl-1,4-naphthoquinone (DMNQ) and 1,4-naphthoquinone (NQ), were examined. These quinones differ in their arylation capacity (NQ > menadione ≫ DMNQ) and in their potential to induce redox cycling (NQ ≈ menadione ≫ DMNQ). The glutathione status (reduced and oxidized glutathione) of the hepatocytes was determined using HPLC after derivatization with monobromobimane. Protein thiols were measured spectrophotometrically and the energy charge of the cells was determined with HPLC using ion pair chromatography. The leakage of lactate dehydrogenase was used as a marker for cell viability. All three quinones caused alterations of the glutathione status of the exposed cells but the effects were markedly different. Exposure to DMNQ resulted in a slow decrease of reduced glutathione and an increase of mixed disulfides. The other two quinones caused an almost complete depletion of reduced glutathione within 5 min. Hepatocytes exposed to NQ accumulated oxidized glutathione whereas menadione-exposed hepatocytes showed increased levels of mixed disulfides. We did not find any effects of DMNQ (200 μM) on protein thiols, energy charge or cell viability. There was a clear difference in the effects of menadione and NQ on protein thiols, energy charge and cell viability: exposure to NQ resulted in a more extensive decrease of protein thiols and energy charge and an earlier onset of lactate dehydrogenase leakage. From our results we conclude that the arylation capacity of a quinone is a determining factor in the cytotoxic potential of such compounds and that the decrease of protein thiols and of the energy charge are critical events preceding loss of cell viability.

Hepatic cytochrome P450 induction in goats: Effects of model inducers on the metabolism of alkoxyresorufins, testosterone and ethylmorphine, and on apoprotein and mRNA levels

Male and female African dwarf goats were treated orally with phenobarbital (PB) or triacetyloleandomycin (TAO), or subcutaneously with beta-naphthoflavone (BNF). Hepatic microsomal cytochrome P450 content was increased by PB and TAO, but not by BNF. PB effects on P450 activities were non-selective: ethoxyresorufin deethylase (EROD) and pentoxyresorufin depentylase (PROD), hydroxylation of testosterone (TST) and demethylation of ethylmorphine (ETM) were all induced by a factor of 2-3. A similar non-selective induction was observed with TAO, except for EROD and PROD (no effects). After PB and TAO treatment, increased levels of a protein cross-reactive with anti-sheep P450 3A and 2B were found. Thus, in dwarf goats, both PB and TAO appeared to be P450 3A inducers. Selective PB effects related to a P450 2B form on PROD are lacking but 16 alpha-hydroxylation of TST was induced markedly. At the mRNA level, PB induced an mRNA that showed good sequence homology with a human P450 3A4 cDNA probe, rather than with a rat 3A1 probe. BNF selectively induced EROD, whereas TST hydroxylation and ETM dealkylation were inhibited. With BNF-treated animals, increased concentrations of a protein cross-reactive with anti-rat P450 1A1/1A2 and of an mRNA that showed homology with a human 1A1 cDNA probe, but not with a mouse 1A1/1A2 probe, were observed.

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.

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.