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Styrene-7,8-oxide (SO), the main intennediate metabolite of styrene, induces hyperkeratosis and tumors in the forestomach of rats and mice upon chronic administration by gavage. The aim of this study was to investigate wbether DNA binding could be responsible for the carcinogenic effect observed. [7-\(^3\)H]SO was administered by oral gavage in com oll to male CD rats at two dose levels (1.65 or 240 mg/kg). After 4 or 24 h, forestomach, glandular stomach and Uver were exclsed, DNA was isolated and its radioactivity detennined. At the 4 h time polnt, the DNA radioactivity was below the Iimit of detection in the torestornach and the liver. Expressed in the units of the covalent bindlng Index, CBI = (pmol adduct/mol DNA nucleotide)/(mmol cbemical administeredlkg body wt), the DNA-binding potency was below 2.6 and 2.0 respectively. In the glandular stomach at 4 b, and in most 24 b samples, DNA was slightly radiolabeled. Enzymatic degradation of the DNA and separation by HPLC ofthe normal nucleotides sbowed that the DNA rad.ioactivity represented biosynthetic incorporation of radlolabel into newly synthesized DNA. The Iimit of detection of DNA adducts in the glandular stomach was 1.0. In a second experlment, [7-\(^3\)H]SO was administered by i.p. injection to male 86C3Fl rnice. Liver DNA was analyzed after 2 h. No radloactivity was detectable at a Iimit of detection of CBI < 0.6. In agreement with the relatively long half-life of SO in animals, the cbemical reactivity of SO appears to be too low to result in a detectable production of DNA adducts in an in vivo situation. Upon comparison with the DNA-binding of other carcinogens, a purely genotoxic mechanism of tumorigenJc action of SO is unlikely. The observed tumorigenic potency in the forestomach could be the result of strong tumor promotion by high-dose cytotoxicity foUowed by regenerative hyperplasia.
(6,7-\(^3\)H] Estrone (E) and [6,7-\(^3\)H]estradiol-17ß (E\(_2\)) have been synthesized by reduction of 6-dehydroestrone and 6-dehydroestradiol with tritium gas. Tritiated E and E\(_2\) were administered by oral gavage to female rats and to male and female hamsters on a dose level of about 300 \(\mu\)g/kg (54 mCi/kg). After 8 h, the liver was excised from the rats; liver and kidneys were taken from the hamsters. DNA was purified either directly from an organ homogenate or via chromatin. The radioactivity in the DNA was expressed in the units of the Covalent Binding Index, CBI = (\(\mu\)mol chemical bound per mol Similar considerations can be made for the liver where any true covalent DNA binding must be below a Ievel of 0.01. It is concluded that an observable tumor induction by estrone or estradiol is unlikely to be due to DNA binding. DNA-P)/(mmol chemical administered per kg b.w.). Rat liver DNA isolated via chromatin exhibited the very low values of 0.08 and 0.09 for E and E\(_2\) respectively. The respective figures in hamster liver were 0.08 and 0.11 in females and 0.21 and 0.18 in the males. DNA isolated from the kidney revealed a detectable radioactivity only in the female, with values of 0.03 and 0.05 for E and E\(_2\) respectively. The values for male hamster kidney were < 0.01 for both hormones. The minute radioactivity detectable in the DNA samples does not represent covalent binding to DNA, however, as indicated by' two sets of control experiments. (A) Analysis by HPLC of the nucleosides prepared by enzyme digest of liver DNA isolated directly or via chromatin did not reveal any consistent peak which could have been attributed to a nucleoside-steroid adduct. (B) All DNA radioactivity could be due to protein contaminations, because the specific activity of chromatin protein was determined to be more than 3 ,000 tim es high er than of DNA. The high affinity of the hormone to protein was also demonstrated by in vitro incubations, where it could be shown that the specific activity of DNA and protein was essentially proportional to the concentration of radiolabelled hormone in the organ homogenate, regardless of whether the animal was treated or whether the hormone was added in vitro to the homogenate. Carcinogens acting by covalent DNA binding can be classified according to potency on the basis of the Covalent Binding Index. Values of 10\(^3\)-10\(^4\) have been found for potent, 10\(^2\) for moderate, and 1-10 for weak carcinogens. Since estrone is moderately carcinogenic for the kidney of the male hamster, a CBI of about 100 would be expected. The actually measured Iimit of detection of 0.01 places covalent DNA binding among the highly unlikely mechanisms of action.
In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A\(_2\) receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cycla.se, and platelet aggregation studies. Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited by compounds 2a-d with K\(_i\) values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affmity binding at solubilized A\(_2\) receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [\(^3\)H]DPCPX and for the agonist [\(^3\)H]CCPA gave K\(_i\) values in the nanomolar range, and the compounds showed moderate A\(_2\) selectivity. In order to improve this selectivity, the correaponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. A\(_1\) expected, the 5'-N-ethyluronamide derivatives retained the A\(_2\) affinity whereas the A\(_1\) affinity was attenuated, resulting in an up to 10-fold increase in A\(_2\) selectivity. A similar patternwas observed in adenylate cyclase assays andin platelet aggregation studies. A 30- to 45-fold selectivity for platelet A\(_2\) receptors compared to A\(_1\) receptors was found for compounds 8a-c in adenylate cyclase studies.
Adenosine receptor agonists: Synthesis and biological evaluation of 1-deaza analogues of adenosine
(1988)
In a search for more selective A\(_1\) adenosine receptor agonists, N\(^6\)-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N\(^6\)-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N\(^6\)-cyclohexyl-l-deazaadenosine (1-deazaCHA, Sc), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-ß-D-ribofuranosyl-3Himidazo[ 4,5-b]pyridine (1). On the other band, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-ß-D-ribofuranuronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-ß-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine (4), in an attempt to find a more selective A\(_2\) agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase andin radioligand binding studies. 1-DeazaNECA (10) proved tobe a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N\(^6\)-substituted 1-deazaadenosines largely retain the A\(_1\) agonist activity of their parent compounds, but lose some of their A\(_2\) agonist activity. This results in A\(_1\)-selective compounds, of which N\(^6\)cyclopentyl- 2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A\(_1\) adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A\(_1\) receptor mediated adenosine actions.
The aim of this study was to determine whether o-chlorobenzylidene malononitrile ( CS) exhibits any genotoxic activity towards Salmonella or mammalian DNA in vivo. CS was synthesized with a [\(^{14}\)C]-label at the benzylic carbon atom. It was administered i. p. at a dose level of 13 mg/kg (1 mCi/kg) to young adult male rats. Liverand kidney DNA was isolated after 8, 25, and 75 h. The radioactivity was at (liver, 8 and 75 h) or below (all other samples) the limit of detection of 3 dpm. Therefore, a possible binding of CS to DNA is at least 10\(^5\) times lower than that of the strong hepatocarcinogen aflatoxin B1, and 4,000 times lower than that of vinyl chloride. In contrast to this lack of DNA binding, but in agreement with the chemical reactivity of CS, a binding to nuclear proteins could be detected with specific activities ranging between 50 and 121 dpm/mg for liver and between 3 and 41 dpm/mg for kidney. Protein binding could well be responsible for its pronounced cytotoxic effects. Cs was also tested in the Ames Salmonella/microsome assay. Strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were used with or without pre-incubation. Only with strain TA 100 and only without pre-incubation, a doubling of the number of revertants was detectable at the highest dose Ievels used, 1,000 and 2,000 !lg CS per plate. With pre-incubation of TA 100 with CS, a slight increase of the number of revertants was seen at 100 and 500 !lg per plate, and a subsequent fall below control values at 1,000 J.tg. A check for the number of surviving bacteria revealed a strong bacteriotoxicity of the higher doses of es so that the calculated mutation frequencies, i.e., the oumber of revertants per number of surviving bacteria, increased with doses up to 500 !J.g. This toxicity could be counteracted in part by the addition of increasing amounts of rat liver microsomes. In the view of these results, and taking into account the rare and low exposure of man, it is concluded that CS will not create a risk for the induction of point mutations or of carcinogenic processes mediated by DNA binding.
Investigation of the Potential for Binding of Di(2-ethylhexyl) Phthalate (DEHP) and Di(2- ethylhexyl) Adipate (DEHA) to Liver DNA in Vivo. VON DÄNIKEN, A., LUTZ, W. K., JÄCKH, R., AND ScHLATTER, C. (1984). Toxico/. App/. Pharmaco/. 73, 373-387. It was the aim oftbis investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or witbout pretreatment for 4 weeks with 1% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [\(^{14}\)H]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The Iimit of detection, CBI < 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [\(^{14}\)C]- and [\(^{3}\)H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where tbe carcinogenmodified nucleoside or base adducts are expected. The respective Iimits of detection were at 0.07 and 0.04 CBI units for the \(^{14}\)C and \(^{3}\)H Iabels, respectively. The experiments with [\(^{14}\)C]- and [\(^{3}\)H]DEHA, labeled in the alcobol moiety and administered to mice, revealed aminute radioactivity of <50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which bad been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for \(^{14}\)C and 0.01 to 0.12 for \(^{3}\)H resulted. Determination of the level· of \(^{14}\)C02 expiration revealed a linear correlation with the speciftc activity of DNA. Experiments with 2-ethyl[ 1-\(^{14}\)C]hexanol perfonned with both rats and mice allowed the conclusion tbat most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01. Pretreatment of the animals witb unlabeled compound bad no effect on the DNA radioactivities in either species. The present negative data, in conjunction witb other negative short-term tests for mutagenicity, strongly indicate that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP and DEHA in rodents.
Lack of covalent binding to rat liver DNA of the hypolipidemic drugs clofibrate and fenofibrate
(1981)
\(^{14}\)C-Labelled clofibric acid and fenofibric acid were administered p.o. to 200 g male and female rats. After 10 h, liver nuclear DNA and protein were isolated and the radioactivity was determined. Binding to protein was clearly measurable whereas no binding to DNA could be detected from any drug. A comparison of the Iimit of detection of such DNA binding with well-known chemical carcinogens revealed that the known hepatocarcinogenicity of clofibrate cannot be based upon an initiating, DNA damaging, mode of action but must be due to other, nongenotoxic, mechanisms such as peroxisome proliferation, hepatomegaly, or cytotoxicity due to protein binding. The risk assessment in man and the interpretation of the carcinogenicity data for rodents are discussed.
Neoplastic cell transfonnation induced by estrogens and some other carcinogen& such as benzene appears to involve the induction of mitotic aneuploidy rather than DNA damage and point mutations. As metabolic activation may also play an important roJe in the mechanism of carcinogenesis of these nongenotoxic compounds, we have studied the Interaction of reactive quinone metabolites of various estrogens and of benzene with the major microtubular protein, tubulin, in a cell-free system. Covalent binding of the radioactively labeled metabolites to the a- and 13-subunit of tubulin was found to depend on the structure of the metabolite. When the adducted tubulins were tested in vitro for their ability to polymerize to microtubules, Inhibition of microtubule assembly was obsened in every case, although to varying extents. It is proposed that the fonnation of covalent tubulin adducts may impair the formation of mitotic spindies and thus contribute to chromosomal nondisjunction and aneuploidy induction.
'lbe mouse skin tumor model was used to investigate whether the Ievel of DNA 8dducts and/or the rate of cell division in the epidermis are indicators of the risk of cancer formation for an individual in an outbred animal popul8tion. A high risk was considered to be reftected by 8 short latency period for the 8ppearance of 8 papilloma. Fernale NMRI mice were treated twice weekly with 2.5 nmol 7 ,12-dimethylbenz[a]antbracene (DMBA) and 3 nmoi12-0-tetradecanoylphorbol-13- 8cetate (TPA) and the appearance of papillomas was registered. The first papilloma 8ppeared after 7.5 weeks. After 17 weeks, when 12 of 14 mice bad 8t least one papilloma, an osmotic minipump deliverlog 5-bromo-2'deoxyuridine (BrdU) was implanted into eacb mouse for 24 h. The mice were killed after 24 h ~d the epidermis was analyzed for D:MBA-nucleotide 8dducts by 32p.postlabeling, for the cell number per unit skin length, and for the labeling index for DNA synthesls. Unexpectedly, D:MBA-nucleotide 8dduct Ievels were highest in those anima1s wbich showed the Iongest latency periods. Adduct Ievels were negatively correlated with the 18beling index, indicating that dilution of adducts by cell division was a predominant factor in determining average adduct concentrations. Individual tumor-latency time was not corTelated with either cell ntunber or labeling index. This could be due to the fact that the measurements only provided 8veraged data and gave no infonnation on the specific situation in clones of premalignant cells. Under the conditions of tbis assay, therefore, neither DNA adduct Ievels nor information on the average kinetics of cell division bad a predidive value for the individual amcer risk withln a group of outbred animals receiving the same treatment
Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin’s pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20–40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin’s lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.
Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[ (3-cholamidopropy l)dimethylammonio ]- 1-propanesulfonic acid (CHAPS). In membrane fragmentsandsoluble extracts neuropeptide Y bindingwas time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective Kn and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y bindingwas specifically inhibited by the nonhydrolyzable GTP analog guanosine 5' -0- (3-thiotripbosphate) in a concentration-dependent manner, with IC\(_{50}\) values of 28 and 0.14 \(\mu\)M for membrane- bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. CrossHoking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.
A\(_1\) adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaflinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1 ,3-[\(^3\)H]dipropyl-xanthine ([\(^3\)H]DPCPX) gave a Kdvalue of 0.7 nM and a Bmax value of 82± 13 fmol/mg protein. For the highly A\(_1\)-selective agonist 2-chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [\(^3\)H]DPCPX binding gave a pharmacological profile with R-N\(^6\)-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCP A to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N\(^6\)- \(^{125}\)I-p-hydroxyphenylisopropyladenosine ([\(^{125}\)I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A\(_1\) receptors labelled in brain membranes. Therefore, coated vesicles contain A\(_1\) adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A\(_1\) receptors in these vesicles are not a processed receptor fonn. These results confirm that A\(_1\) receptors in coated vesicles are coupled to a G-protein, and it appears that the A\(_1\) receptor systems in coated vesicles andin plasma membranes are identical.
To date, all risk assessment studies on benzene have been based almost exclusively on epiderniological data. Wehave attempted a more integrated and quantitative evaluation of carcinogenic risk for hurnans, trying to utilize, in addition to the epidemiological data, all data available, specifically data on metabolism, genotoxicity, and carcinogenicity in small rodents. An integrated evaluation of the globality of the available data seems to suggest a progressive saturation of metabolic capacity both for man and rodents between 10 and 100 ppm. The most susceptible target cells seem tobe different in humans (predominant induction of myelogenous leukemia) and small rodents (induction of a wide variety of tumors). Nevertheless, both epidemiological and experimental carcinogenicity data tend to indicate a flattening ofthe response for the highest dosages, again suggesting a general Saturation of mechanisms of metabolic activation, extended to different target tissues. From a quantitative point of view, the data suggest a carcinogenic potency at 10 ppm two to three times higher than that computable by a linear extrapolation from data in the 100 ppm range. These observations are in accord with the recent proposal of the European Economic Community of reducing benzene time-weighted average occupationallevels from 10 to 5 ppm.
Male rats were treated for 2 months with 1000 ppm nafenopin in the diet or for 4 or 7 days with a choline-devoid low-methionine diet. DNA was isolated from the livers and analyzed for the presence of cis-thymidine glycol-3'-phosphate (cis-dTGp) by 32P-postlabeling and for the Ievel of 8-hydroxy-deoxyguanosine (8-0H-dG) by electrochemical detection (ECD). In no DNA sample was the Ievel of cis-dTGp above the Iimit of detection of 1 modified thymidine per 106 nucleotides. With 8-0H-dG, a background Ievel of this modification of 20 8-0H-dG per 106 nucleosides was found in liver DNA of control rats, which was not affected by either treatment. It is postulated for thymidine glycol that a potential increase was below the Iimit of detection or was rapidly repaired in vivo and that the steady-state Ievel of endogenous 8-hydroxydeoxyguanosine appears not tobe influenced by the treatments chosen.
Terahertz electromagnetic fields are non-ionizing electromagnetic fields in the frequency range from 0.1 to 10 THz. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along human exposure to these fields. Up to now, only a limited number of investigations on biological effects of terahertz electromagnetic fields have been performed. Therefore, research is strongly needed to enable reliable risk assessment. Cells were exposed for 2 h, 8 h, and 24 h with different power intensities ranging from 0.04 mW/cm2 to 2 mW/cm2, representing levels below, at, and above current safety limits. Genomic damage on the chromosomal level was measured as micronucleus formation. DNA strand breaks and alkali-labile sites were quantified with the comet assay. No DNA strand breaks or alkali-labile sites were observed as a consequence of exposure to terahertz electromagnetic fields in the comet assay. The fields did not cause chromosomal damage in the form of micronucleus induction.