A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocyte/DNA repair assay

Methapyrilene and four related antihistamines were evaluated for their ability to cause DNA repair measured autoradiographically as unscheduled DNA synthesis (UDS) in primary cultures of Fischer-344 rat hepatocytes. Methapyrilene failed to induce UDS at all doses tested while pyrilamine and tripelennamine induced a concentration-dependent increase in DNA repair. Doxylamine and thenyldiamine, previously untested in this system, induced a weak response at the highest non-toxic doses tested. Methapyrilene was clearly cytotoxic at doses of 100 microM and higher, as judged by morphology, and precursor incorporation into RNA and protein. Precursor incorporation into RNA was irreversibly inhibited 90% and 55% at 1000 microM and 100 microM methapyrilene, respectively, while precursor incorporation into protein was inhibited 80% and 60%. These data verify the genotoxicity of pyrilamine and tripelennamine and the failure of methapyrilene to elicit DNA repair, and suggest that doxylamine and thenyldiamine may be weak DNA-damaging agents.     

A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocytes/DNA repair assay

Methapyrilene and four related antihistamines were evaluated for their ability to cause DNA repair measured autoradiographically as unschedules DNA synthesis (UDS) in primary cultures of Fischer-344 rat hepatocytes. Methapyrilene failed to induce UDS at all doses tested while pyrilamine and tripelennamine induced a concentration-dependent increase in DNA repair. Doxylamine and thenyldiamine, previously untested in this system, induced a weak response at the highest non-toxic doses tested. Methapyrilene was clearly cytotoxic at doses of 100 μM and higher, as judged by morphology, and precursor incorporation into RNA and protein. Precursor incorporation into RNA was irreversibly inhibited 90% and 55% at 1000 μM and 100 μM methapyrilene, respectively, while precursor incorporation into protein was inhibited 80% and 60%. These data verify the genotoxicity of pyrilamine and tripelennamine and the failure of methapyrilene to elicit DNA repair, and suggest that doxylamine and thenyldiamine may be weak DNA-damaging agents.     

Methapyrilene hepatotoxicity is associated with increased hepatic glutathione, the formation of glucuronide conjugates, and enterohepatic recirculation

The mechanisms by which acute administration of methapyrilene, an H(1)-receptor antihistamine causes periportal necrosis to rats are unknown. This study investigated the role of the hepato-biliary system in methapyrilene hepatotoxicity following daily administration of 150 mg/kg per day over 3 consecutive days. Biliary metabolites of methapyrilene were tentatively identified. In male Han Wistar rats administration of methapyrilene significantly increased hepatic reduced glutathione (GSH) to 140% of control levels 24 h following the last dose. There were no significant changes in the activities of glutathione-related enzymes, glutathione peroxidase (GPx) and reductase (GSH), glutathione S-transferase (GST), and gamma-glutamyl cysteine synthetase (gamma-GCS) over 3 days of methapyrilene administration. Methapyrilene treatment resulted in no significant increase in excretion of biliary oxidized glutathione (GSSG), a sensitive marker of oxidative stress in vivo, following the third dose. [3H]Methapyrilene-derived radioactivity was detected in bile, to a greater extent than in feces, indicating that methapyrilene and/or metabolites underwent enterohepatic recirculation. Cannulation and exteriorization of the bile duct (to interrupt enterohepatic recirculation) afforded some protection against the hepatotoxicity, assessed by clinical chemistry and histopathology. Liquid chromatography-mass spectrometry (LC-MS) analysis of bile indicated the presence of unmetabolized methapyrilene, methapyrilene O-glucuronide and desmethyl methapyrilene O-glucuronide. These data demonstrate that acute methapyrilene hepatotoxicity in vivo is not a consequence of GSH depletion, or oxidative stress, but that enterohepatic recirculation of biliary metabolites may be important. Progressive exposure to non-oxidizing, reactive metabolic intermediates may be responsible for hepatotoxicity.     

Genotoxicity,toxicity, and carcinogenicity of the antihistamine methapyrilene (MTR 07216)

The antihistamine methapyrilene hydrochloride (MP) has been shown to be a potent hepatocarcinogen in rats, but not in hamsters or guinea pigs. This finding is in contrast to the relative nongenotoxicity of this compound. MP has been evaluated for genotoxicity in a wide variety of short-term tests and has generally demonstrated little genotoxic activity. One exception to this is the mouse lymphoma L5178Y mutagenesis assay, in which MP produced a very significant increase in small colony mutants with concomitant chromosomal damage in these cells. MP also induced positive responses in several cell transformation assays. One potentially very significant effect of MP is that it induces a large increase in hepatic cell proliferation coupled with mitochondrial proliferation in the livers of treated animals. This effect is discussed as a possible mechanism of liver tumor induction in rats.     

Genotoxicity, toxicity, and carcinogenicity of the antihistamine methapyrilene

The antihistamine methapyrilene hydrochloride (MP) has been shown to be a potent hepatocarcinogen in rats, but not in hamsters or guinea pigs. This finding is in contrast to the relative nongenotoxicity of this compound. MP has been evaluated for genotoxicity in a wide variety of short-term tests and has generally demonstrated little genotoxic activity. One exception to this is the mouse lymphoma L5178Y mutagenesis assay, in which MP produced a very significant increase in small colony mutants with concomitant chromosomal damage in these cells. MP also induced positive responses in several cell transformation assays. One potentially very significant effect of MP is that it induces a large increase in hepatic cell proliferation coupled with mitochondrial proliferation in the livers of treated animals. This effect is discussed as a possible mechanism of liver tumor induction in rats.     

Systems toxicology: integrated genomic, proteomic and metabonomic analysis of methapyrilene induced hepatotoxicity in the rat

Administration of high doses of the histamine antagonist methapyrilene to rats causes periportal liver necrosis. The mechanism of toxicity is ill-defined and here we have utilized an integrated systems approach to understanding the toxic mechanisms by combining proteomics, metabonomics by 1H NMR spectroscopy and genomics by microarray gene expression profiling. Male rats were dosed with methapyrilene for 3 days at 150 mg/kg/day, which was sufficient to induce liver necrosis, or a subtoxic dose of 50 mg/kg/day. Urine was collected over 24 h each day, while blood and liver tissues were obtained at 2 h after the final dose. The resulting data further define the changes that occur in signal transduction and metabolic pathways during methapyrilene hepatotoxicity, revealing modification of expression levels of genes and proteins associated with oxidative stress and a change in energy usage that is reflected in both gene/protein expression patterns and metabolites. The difficulties of combining and interpreting multiomic data are considered.     

Altered distribution of hexokinase and glucokinase between parenchymal and nonparenchymal cells of rat liver after methapyrilene intoxication

The cell number as well as the hexokinase and glucokinase activity of liver parenchymal and nonparenchymal cells were studied in methapyrilene treated rats. The number of nonparenchymal cells was doubled after treatment with methapyrilene for two weeks while that of hepatocytes remained constant. The hexokinase activity was increased fourfold in the nonparenchymal cell fraction while it was unchanged in the parenchymal cells. The glucokinase activity was decreased in the hepatocytes to one third. Hence, the increased hexokinase activity was due to a proliferation of nonparenchymal cells rather than to a toxic dedifferentiation of hepatocytes.     

Glutathione and lipid peroxide levels in rat liver following administration of methapyrilene and analogs

The possibility was examined that the induction of tumors in rat liver by feeding methapyrilene, which is not mutagenic, is related to effects on glutathione levels and lipid peroxidation. Fischer 344 rats were given single-dose and multiple-dose treatments with the anti-histamine methapyrilene (MP), which is carcinogenic in rats, and with two non-carcinogenic analogs, methafurylene (MF) and thenyldiamine (TD) and the effects on malonaldehyde (MDA) formation and glutathione (GSH) levels in the liver were investigated. After a single dose, MDA levels were increased at 6 h by MF and TD and at 24 h by MP. MDA levels returned to normal after 30 h with MP and MF, but not with TD. Levels of MDA (and other TBA-reactive products) after four daily treatments were most elevated by TD, less elevated by MP, and were lowered by MF. Forty-two hours following treatment with both MP and MF, MDA levels had returned to normal, but in TD-treated animals MDA remained high. GSH levels were highest after MF and MP, and remained high at 42 h, but TD induced only a small increase. There appears to be increased lipid peroxidation in the liver as a result of treatment of rats with MP, MF and TD. The greater response induced by TD, as well as the increased liver GSH levels after repeated administration of all three drugs indicate that lipid peroxidation in rat liver is not a particular effect related to the liver carcinogen methapyrilene.     

Involvement of lipoxygenase products in myometrial contractions

Studies with an in situ preparation of guinea pig uterus suggest the possible involvement of the lipoxygenase pathway of arachidonic acid (AA) metabolism in myometrial contractions. Female guinea pigs were sensitized to ovalbumin (OA) on day one of their estrous cycle. On day 14, these pigs were anesthetized and the uterus was cannulated for measuring contractions. OA challenge, with histamine antagonism (methapyrilene) resulted in uterine contractions which significantly raised myometrial tonus, presumably due to AA metabolites. Pretreatment with high doses of indomethacin resulted in only 60% inhibition of the OA induced contraction, suggesting the remaining contraction was due to something other than cyclooxygenase products. In the presence of indomethacin and methapyrilene, the addition of AA to increase available substrate caused increased myometrial tone following antigen challenge. This increase in uterine tone was inhibited in a dose dependent fashion by FPL-55712 demonstrating that leukotrienes can contract the uterus and that antigen challenge may provide a means for studying leukotriene involvement in uterine pathophysiology.     

Methapyrilene: DNA as a possible target

The structural features contributing to the potential carcinogenicity, DNA-reactivity and genotoxicity of methapyrilene and its non-carcinogenic congener pyrilamine were examined. The analyses suggest that the former has the potential for DNA-reactivity, a property which is absent from the latter.     

DNA as a Flocculation Factor in Pseudomonas sp.

A component responsible for flocculation was extracted from Pseudomonas strain C-120 by treating the cells with 3 M guanidine hydrochloride. The guanidine hydrochloride-extracted cells were reflocculated, not only with the guanidine hydrochloride extract but with DNA prepared from various bacteria. The reconstituted flocs were deflocculated by deoxyribonuclease or guanidine hydrochloride which indicated that the reconstituted flocs closely resembled natural flocs. In reconstitution experiments using Escherichia coli DNA at different molecular weights, it was found that DNA with a molecular weight higher than about 6 × 10⁶ was required to flocculate the guanidine hydrochloride-extracted cells. Heat-denatured DNA did not flocculate the guanidine hydrochloride-extracted cells. DNA with a high molecular weight was detected in the guanidine hydrochloride extract. It was concluded that the component involved in flocculation of this organism was highly polymerized double stranded DNA.     

An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity

The Bhas 42 cell transformation assay is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to have acquired an initiated state in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay is capable of detecting both tumor-initiating and tumor-promoting activities of chemical carcinogens. The full assay protocol consists of two components, the initiation assay and the promotion assay, to detect the initiating activity and the promoting activity, respectively. An international study was carried out to validate this cell transformation assay in which six laboratories from three countries participated. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the initiation assay, concordant results were obtained by three laboratories for eight out of ten chemicals and in the promotion assay, concordant results were achieved for ten of twelve chemicals. The positive results were obtained in all three laboratories with the following chemicals: 2-acetylaminofluorene was positive in both initiation and promotion assays; dibenz[a,h]anthracene was positive in the initiation assay; sodium arsenite, lithocholic acid, cadmium chloride, mezerein and methapyrilene hydrochloride were positive in the promotion assay. o-Toluidin hydrochloride was positive in the both assays in two of the three laboratories. d-Mannitol, caffeine and l-ascorbic acid were negative in both assays in all the laboratories, and anthracene was negative in both assays in two of the three laboratories except one laboratory obtaining positive result in the promotion assay. Consequently, the Bhas 42 cell transformation assay correctly discriminated all six carcinogens and two tumor promoters from four non-carcinogens. Thus, the present study demonstrated that the Bhas 42 cell transformation assay is transferable and reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In addition, by comparison of the present results with intra-laboratory data previously published, within-laboratory reproducibility using the Bhas 42 cell transformation assay was also confirmed.     

Acceptor sites for the oestrogen receptor in hen oviduct chromatin.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.     

Structure of Deoxyribonucleic Acid on the Cell Surface During Uptake by Pneumococcus

We exposed competent cells of Diplococcus pneumoniae to high-molecular-weight donor deoxyribonucleate (DNA) and examined the state of the DNA bound to them in forms sensitive to deoxyribonuclease I. The portion elutable with 5 M guanidine hydrochloride was shown to be native, of much lower molecular weight (4 x 10(6) to 5 x 10(6)) than the donor, and as active in further transformation as sheared DNA of the same size. The portion resistant to release by guanidine hydrochloride was also shown to be native and active in transformation. These results, along with previous ones, imply that the breaks produced outside the cell are not at genetically specific sites. Furthermore, it was found that entry past the cell barrier to deoxyribonuclease could occur at 0 C by a process sensitive to ethylenediaminetetraacetate.     

Protein synthesized early after infection is linked to the termini of adenovirus type 2 DNA synthesized in vivo and in vitro.

The human adenovirus DNA genome contains a protein (CBP, or covalently bound protein) linked to each 5' terminus. To assess whether CBP is synthesized early, infected cells were incubated with hydroxyurea from 1 to 18 h postinfection, the hydroxyurea was removed, cycloheximide was added, and viral DNA was labeled with [3H]thymidine from 18 to 23 h postinfection. Removal of hydroxyurea at 18 h postinfection permits the synthesis of viral DNA, whereas cycloheximide maintains the block in late viral protein synthesis. Three lines of evidence are presented to show that viral 3H-labeled DNA prepared by this procedure was linked to CBP: (I) the DNA sedimented more rapidly than protein-free DNA (i.e., protinase treated) in neutral sucrose gradients containing guanidine hydrochloride; (ii) the DNA banded at a lower density than protein-free DNA in CsCl gradients containing guanidine hydrochloride; and (iii) neither the 3H-labeled DNA nor the end fragments produced by EcoRI digestion entered a 1.4% agarose gel during electrophoresis. These experiments are strong evidence that CBP is not a product of a late viral gene and is therefore the product of either an early viral gene or a cell gene. Experiments were performed to test whether CBP is attached to viral DNA synthesized in vitro by a soluble complex that synthesizes exclusively viral DNA as completed viral genomes in vitro. In vitro-labeled DNA was analyzed by velocity sedimentation, equilibrium sedimentation, and agarose gel electrophoresis as described above. Our results indicate that the majority of in vitro-synthesized DNA molecules were attached to CBP. These results, which indicate that CBP is synthesized early after infection and is attached to viral DNA labeled in vitro by a soluble replication complex, are consistent with the idea that CBP may play a role in viral DNA replication.     

Rapid isolation of DNA from Staphylococcus aureus.

We describe a Staphylococcus aureus bulk DNA isolation procedure which uses detergent and guanidine hydrochloride to free the nucleic acid from contaminants. The procedure is rapid and yields high-molecular-weight DNA suitable for molecular biological procedures.     

Rapid isolation of DNA from Staphylococcus aureus

We describe a Staphylococcus aureus bulk DNA isolation procedure which uses detergent and guanidine hydrochloride to free the nucleic acid from contaminants. The procedure is rapid and yields high-molecular-weight DNA suitable for molecular biological procedures.     

New anthracenedione derivatives: Interaction with DNA and biological effects

Two anthracenedione derivatives [1 - (ω - diethylaminopropylamido) - 4 - hydroxy - 9,10 - anthracenedione hydrochloride (I) and 1 - (ω - diethylamino-propylamido) - 2 - methoxy - 4 - hydroxy - 9,10 - anthracenedione hydrochloride (II)], having an electron-rich planar chromophore and an amino-substituted side chain, have been synthesized. Their binding ability to DNA was investigated by means of spectroscopic, equilibrium dialysis and fluorescence measurements. Their inhibition efficiency on nucleic acid synthesis was also evaluated both in mouse and human cells. Our results indicate that, in comparison with adriamycin, compound I shows a slightly weaker complexation ability to DNA, while compound II interacts with DNA at a substantially lower level. These data match quite well with the biological response on the inhibition of DNA and RNA synthesis exhibited by the above mentioned compounds; in fact compound I is slightly less efficient than adriamycin and about ten times more efficient than compound II. The close relationship between the results of physicochemical and biological studies is discussed.     

Direct analysis of microbial extracts containing metabolites of ethylenediamine-type antihistamines via high-performance liquid chromatography-thermospray mass spectrometry

The utility of high-performance liquid chromatography-thermospray mass spectrometry (HPLC-TSMS) for the characterization of the ethylenediamine-type antihistamines, pyrilamine, methapyrilene, tripelennamine, and thenyldiamine, and their methylene chloride-extractable microbial metabolites from a biological matrix is demonstrated. Typically, the [M + H]+ ion was detected as the base peak in the TS mass spectra of these compounds. The ethylenediamine-type antihistamine metabolites were detected in an extract of a fungal culture grown in the presence of 5 mg of the antihistamine. A detection limit of 200 ng was observed for the HPLC-TSMS analysis of pyrilamine.     

Interaction of deoxyribonucleic acid with anthracenedione derivatives

Spectrophotometric, calorimetric and chrioptical techniques have been used to investigate the interaction of two new anthracenedione derivatives, 1-(ω-diethylaminopropylamido)-4-hydroxy-9,10-anthracenedione hydrochloride (I) and 1-(ω)-diethylaminopropylamido)-2-methoxy-4-hydroxy-9,10-anthracenedione hydrochloride (II) to DNA. Measurements were carried out at four different Na⁺ concenetrations. From the dependence of the binding constant on ionic strength the number of ion pairs formed between the ligand and DNA, along with the binding free energy were estimated. Calorimetric measurements show that the binding process is exothermic for both ligands. Experiments carried out with DNA from various sources indicate no marked preference for G-C or A-T binding sites. Compounds I and II increase the T_m for DNA melting by more than 25°C at high drug/base pair ratios. Circular dichroism studies indicate that the structural properties of DNA are substantially affected by the interaction with the above mentioned compounds. All data from these studies are consistent with an intercalative mechanism of binding for the anthracendione derivatives to DNA.