Roland C. Grafström

The isolation of rat intestinal microsomes with stable cytochrome P-450 and their metabolism of benzo(α)pyrene

Abstract A procedure has been developed for the isolation of microsomes from rat intestinal mucosa with stable cytochrome P -450. Preservation of the hemoprotein has been obtained by including trypsin inhibitor, glycerol, and heparin in the homogenization medium. The spectral properties of the hemoprotein from control and phenobarbital (Pb)- and 3-methylcholanthrene (MC)-treated rats were examined. In fed animals, MC given orally resulted in a 30-fold stimulation of benzo(α)pyrene (BP) monooxygenase within 24 h with a simultaneous increase in cytochrome P -450 ( P -448) but had no effect on NADPH-cytochrome c reductase activity. The effect of MC on BP monooxygenase and cytochrome P -450 ( P -448) could be seen as early as 1.5 h after oral administration. Pb treatment increased cytochrome P -450 levels but had no effect on NADPH-cytochrome c reductase activity and comparatively little effect on BP monooxygenase. In fasted animals, MC also produced large increases in BP monooxygenase activity when compared to control animals. At 7 μ m α-naphthoflavone, BP monooxygenase was inhibited 96% in microsomes from MC-treated rats and stimulated 4.5-fold in microsomes from control animals. The pattern of BP metabolites was similar for intestinal microsomes from control and MC-treated rats but differed sharply from that produced by hepatic microsomes. The 4,5-oxide of BP constituted one of the major intestinal metabolites with only small amounts of dihydrodiols being formed after a 5-min incubation. It is concluded that the cytochrome P -450-linked monooxygenase system present in the intestinal mucosa differs markedly from the hepatic system with regard to induction properties, substrate specificity, and pattern of BP metabolites.

High-Throughput Cell-Based Screening of 4910 Known Drugs and Drug-like Small Molecules Identifies Disulfiram as an Inhibitor of Prostate Cancer Cell Growth

Purpose: To identify novel therapeutic opportunities for patients with prostate cancer, we applied high-throughput screening to systematically explore most currently marketed drugs and drug-like molecules for their efficacy against a panel of prostate cancer cells. Experimental Design: We carried out a high-throughput cell-based screening with proliferation as a primary end-point using a library of 4,910 drug-like small molecule compounds in four prostate cancer (VCaP, LNCaP, DU 145, and PC-3) and two nonmalignant prostate epithelial cell lines (RWPE-1 and EP156T). The EC50 values were determined for each cell type to identify cancer selective compounds. The in vivo effect of disulfiram (DSF) was studied in VCaP cell xenografts, and gene microarray and combinatorial studies with copper or zinc were done in vitro for mechanistic exploration. Results: Most of the effective compounds, including antineoplastic agents, were nonselective and found to inhibit both cancer and control cells in equal amounts. In contrast, histone deacetylase inhibitor trichostatin A, thiram, DSF, and monensin were identified as selective antineoplastic agents that inhibited VCaP and LNCaP cell proliferation at nanomolar concentrations. DSF reduced tumor growth in vivo, induced metallothionein expression, and reduced DNA replication by downregulating MCM mRNA expression. The effect of DSF was potentiated by copper in vitro. Conclusions: We identified three novel cancer-selective growth inhibitory compounds for human prostate cancer cells among marketed drugs. We then validated DSF as a potential prostate cancer therapeutic agent. These kinds of pharmacologically well-known molecules can be readily translated to in vivo preclinical studies and clinical trials. (Clin Cancer Res 2009;15(19):6070–8)

Mutagenesis of xeroderma pigmentosum fibroblasts by acrolein

Acrolein, a short-chain aldehyde encountered as a component of tobacco smoke and as a ubiquitous environmental contaminant, was tested for its toxic and mutagenic effects toward human fibroblast cells. We found that human cells characterized by a deficiency in DNA repair (cells from xeroderma pigmentosum (XP) patients) were much more sensitive (D37 approximately equal to 0.25 microM) to the cytotoxic effects of acrolein than were cells from normal individuals (D37 approximately equal to 0.8 microM). Acrolein was also strongly mutagenic to the XP cells (a dose response was observed between 0.2 and 0.8 microM acrolein); however acrolein did not induce an increase in the mutant frequency of normal fibroblasts. Possible reasons for this apparent lack of mutagenicity in normal human cells are discussed.

Formation of DNA adducts in human buccal epithelial cells exposed to acetaldehyde and methylglyoxal in vitro

Acetaldehyde (AA) and methylglyoxal (MG) are reactive, ubiquitous aldehydes, present in the environment and endogenously formed in animals and humans. They have both been shown to readily form DNA adducts under simulated physiological conditions. We report here on the use of cultured normal and SV40T antigen-immortalized human buccal epithelial cells as model systems for aldehyde exposure of the oral epithelium, occurring through the ingestion of alcoholic beverages and brewed coffee, as well as by inhalation of tobacco smoke and automobile exhaust. By the application of recently developed 32P-postlabeling methods, the presence of both endogenous and induced AA and MG DNA adducts was demonstrated in cultured human epithelial cells. Furthermore, these DNA adducts were formed in a dose-dependent manner at aldehyde concentrations that were relatively nontoxic to the cells.

Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors.

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.

Paracetamol metabolism in the isolated perfused rat liver with further metabolism of a biliary paracetamol conjugate by the small intestine

Abstract The isolated, perfused rat liver metabolized paracetamol to glucuronide. sulphate and glutathione conjugates. Sulphate conjugation was the preferred route of metaholism at the drug concentrations studied, but formation of glutathione conjugate became increasingly prominent at higher paracetamol concentrations. Sulphate conjugation was saturated at a paracetamol concentration in the perfusatc of 5 mM. while formation of glucuronide or glutathione conjugates was not yet saturated at 10 mM. The sulphate conjugate was predominantly excreted into the perfusate whereas the excretion pattern for the glucuronide and glutathione conjugates changed with time. Initially, both these conjugates were almost exclusively excreted in the bile, after 90 min perfusion mainly into the perfusate. Preformed paracetamol conjugates were not transferred from perfusate to bile by the isolated perfused liver. Freshly isolated intestinal cells rapidly converted paracetamol-S-glutathione to paracetamol-S-cysteine which was slowly acetylated to the,N-acetylcysteine derivative. Methionine stimulated, and serine-borate inhibited, the breakdown of paracetamol-S-glutathione by intestinal cells, indicating the involvement of γ-glutamyltranspeptidase in the reaction. Biliary paracctamol-S-glutathione was metabolized similarly by the small intestine in situ; the subsequent appearance of the cysteine conjugate in plasma revealed that breakdown of the glutathione conjugate occurred before or during passage through the intestinal wall. Direct absorption of paracetamol-S-cysteine from the intestinal lumen to the portal blood was verified by instillation of this derivative in the intestinal lumen in situ. Analysis of plasma also indicated enterohepatic circulation and reabsorption of biliary paracetamol and paracetamol glucuronide from the intestinal lumen.

Expression of keratins in normal, immortalized and malignant oral epithelia in organotypic culture

Keratins have been extensively studied in tissues and cultured keratinocytes but limited information is available on epithelia reconstructed in vitro. The aim of this study was to examine keratin expression in organotypic epithelia with normal (NOK), immortalized (SVpgC2a) and malignant (SqCC/Y1) human buccal cells. Organotypic epithelia were derived from 10 days of culture at the air-liquid interface of collagen gels containing human oral fibroblasts using a standardized serum-free medium. Sections were stained immunohistochemically with selected mono-specific antibodies to a range of keratins. Organotypic epithelia showed sharp differences in keratin expression and distribution. K4/K13, K1/K10, K6/K16 were variably expressed in NOK and SqCC/Y1 but were not detected in SVpgC2a. K5 was expressed in all organotypic epithelia but K14 was absent in SVpgC2a. K7 and K8 showed variable expression while K18 was expressed uniformly in all epithelia. K19 was expressed consistently in NOK and K20 was distributed heterogeneously in SVpgC2a. Overall, organotypic cultures of normal keratinocytes express many of the same keratins as buccal mucosa. Further, the loss of keratins in SVpgC2a and their retention in SqCC/Y1 have several features in common with the respective keratin profile of oral epithelial dysplasia and well-differentiated oral squamous cell carcinoma. Although qualitative and quantitative differences exist compared to keratin expression in vivo, these cell lines in organotypic culture may serve in studies of the multi-step progression of oral cancer.

Benzo(α)pyrene metabolism by isolated rat intestinal epithelial cells

Abstract Metabolically active intestinal epithelial cells were isolated using collagenase plus hyaluronidase. Oxygen consumption was measured and was found to be inhibited by KCN, antimycin A, and rotenone. Cells from 3-methylcholanthrene(MC)-treated rats metabolized benzo(α)pyrene (BP) at a rate that was 30-fold greater than control cells. The addition of salicylamide to the incubation medium inhibited conjugation of BP metabolites and facilitated the accumulation of fluorescent and ethylacetate extractable metabolites. Metyrapone, SKF 525-A, α-naphthoflavone (α-NF), and rotenone inhibited BP-metabolism in intestinal cells from MC-treated rats, with α-NF being the most inhibitory. In intestinal cells from control animals, metyrapone and SKF 525-A both inhibited BP metabolism, while α-NF and rotenone both produced an increase in the formation of fluorescent BP products. The distribution of metabolites from MC-treated rats was determined by high-pressure liquid chromatography and compared with authentic BP derivatives. Incubations were conducted for 5 and 30 min in the presence and absence of salicylamide, and 30-min samples incubated in the absence of salicylamide were hydrolyzed with β-glucuronidase or aryl sulfatase. In the absence of salicylamide, large amounts of conjugates were formed, the formation of which were inhibited by salicylamide addition. A product corresponding to the 4,5-oxide constituted the major metabolite after a 5-min incubation, while little dihydrodiol formation occurred. Large amounts of phenolic BP derivatives were also formed. After 30-min incubations, the percentage of products corresponding to the 4,5-oxide decreased, and an increase in dihydrodiol formation was observed. The slow metabolism of the 4,5-oxide and the slow accumulation of dihydrodiols is due to the presence of low epoxide hydrase activity in the intestine. Intestinal cells are capable of xenobiotic metabolism, and offer a convenient method of studying intestinal drug metabolizing processes which may significantly contribute to the overall xenobiotic metabolis in the body.

In vitro studies of aldehyde effects related to human respiratory carcinogenesis

Aldehydes are ubiquitous compounds which are generated from many both endogenous and exogenous sources. Primarily because certain aldehydes are respiratory toxicants and carcinogens in laboratory animals, and also because they are present in both tobacco smoke and automotive emissions, cultured human bronchial cells have been used to study the ability of aldehydes, i.e., acrolein and formaldehyde, to cause pathobiological effects associated with carcinogenesis. Comparative studies indicate that each aldehyde distinctly affects several molecular and cellular variables including colony-forming efficiency, clonal growth rate, membrane integrity, formation of cross-linked envelopes, levels of cytosolic free calcium, low-molecular-weight thiol status, DNA structure, i.e., formation of DNA single-strand breaks and DNA-protein cross-links, and various DNA repair mechanisms. In relation to the toxicity exerted by these agents, acrolein induces differentiation more readily than formaldehyde whereas formaldehyde causes much higher levels of genetic damage than acrolein. However, for all biological endpoints measured, acrolein on a molar basis is always more potent than formaldehyde. Taken together, a variety of effects that relate to cell death, accelerated epithelial terminal differentiation and genotoxicity are associated with aldehyde exposure, which in human airways may have a role in the pathogenesis of various diseases. In the development of cancer, the possible contribution of aldehydes from both intra- and extra-cellular sources may partly depend on the ability of target cells to detoxify and counteract those aldehyde-related effects believed to critically relate to multi-stage carcinogenesis.

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.