The use of short-term tests to measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds

The effect of reducing agents on the nitrosation of methylguanidine (MG) and on the in vitro activation of dimethylnitrosamine (DMN) was examined by measuring DNA-repair synthesis (unscheduled incorporation of [³H]TdR), shifts in alkaline sucrose gradients, frequency of chromosome aberrations, and clone-forming capacity of cultured human fibroblasts. The reducing agents examined were sodium ascorbate, cysteine, cysteamine, and propyl gallate. Since the short-term bioassays used can be quantitated, it has become relatively easy to detect the inhibitory action of reducing compounds on the nitrosation reaction of MG and metabolic activation (with S-9 preparation) of the precarcinogen DMN, to measure their effective dose range, and to establish the most effective ratios between inhibitory agent and reactant. The results indicate that DNA-repair synthesis is a suitable short-term test for studying the numerous combinations and permutations between several carcinogenic or non-carcinogenic agents, and for estimating the capacity of inhibitory agents to affect formation and activation of chemical carcinogens.     

Comparison of the continuous rat hepatoma cell line 2sFou with primary rat hepatocyte cultures for the induction of DNA repair synthesis by nitrosamines, benzo[a]pyrene and hydroxyurea

We have examined the suitability of the continuous rat hepatoma cell line 2sFou for testing the genotoxicity of chemicals in comparison with that of primary rat hepatocyte cultures (HPC). The capacity of the cells for metabolic activation was assessed by measuring induction of DNA-repair synthesis and inhibition of replicative DNA synthesis by the test compounds dimethylnitrosamine (DMN), diethylnitrosamine (DEN), hydroxyurea (HU) and benzo[a]pyrene (BaP), which are substrates for major hepatic and extrahepatic forms of cytochrome P-450 dependent monooxygenases. The cellular capacity for DNA-repair synthesis was assessed using UV-light as a DNA-damaging agent. Repair-specific incorporation of [3H]deoxycytidine (3H-dCyd) caused by UV-light was higher in 2sFou cells than in HPC. In contrast, background repair incorporation of 3H-dCyd in 2sFou cells was only 1/3 that found in HPC. All the test agents induced DNA repair and inhibited DNA synthesis in both 2sFou cells and HPC. The two nitrosamines were more effective in HPC than in 2sFou cells. HU and BaP affected DNA repair and DNA synthesis in the two cell systems at a similar range of concentrations. In general, DNA repair in the 2sFou cells increased near linearly with the concentrations of the test compounds. The data indicate that 2sFou cells are capable of activating hepatotropic pro-mutagens/carcinogens such as dialkylnitrosamines, and are sensitive indicators of DNA damage. In contrast, BaP, a non-hepatotoxic compound, caused only little DNA repair in these cells. Thus, continuously growing cells, such as 2sFou, show a qualitatively similar response to genotoxic chemicals as HPC and offer a potential alternative to HPC for genotoxicity testing.     

Repair synthesis and sedimentation analysis of DNA of human cells exposed to dimethylnitrosamine and activated dimethylnitrosamine

The capacity of dimethylnitrosamine(DMN), and of DMN activated by a NADPH-fortified mouse liver microsomal preparation, to elicit DNA alterations in cultured human fibroblasts was examined. A maximum induction of DNA repair synthesis, estimated by unscheduled incorporation of tritiated thymidine, occurred following 60-minute incubation of the human cells with DMN activated by a NADPH-fortified mouse liver microsomal preparation. A low level of DNA repair activity followed exposure to DMN alone, or to DMN mixed with the microsomal preparation without NADPH or without O₂. The extent of DNA damage, estimated by velocity sedimentation of DNA through alkaline sucrose gradients, was maximum following treatment with DMN mixed with the NADPH-fortified microsomal preparation. The combined application of $\text{in vitro}$ activation systems and estimation of DNA repair synthesis in cultured cells may be exploited in the detection of precarcinogens.     

DNA damage, DNA repair and chromosome aberrations of xeroderma pigmentosum cells and controls following exposure to nitrosation products of methylguanidine

Nitrosation of methylguanidine (MG) led to products that caused DNA fragmentation (shift in sedimentation profiles of velocity centrifugation through alkaline sucrose gradients), a DNA repair synthesis (unscheduled uptake of (³H]TdR), chromosome aberrations and a lethal effect of cultured human fibroblasts. The response of repair-deficient xeroderma pigmentosum cells did not differ from that of controls. The nitrosation of MG must be carried out at a pH level below 3, in order to obtain products that react with cellular DNA. The results show that a DNA repair synthesis of human fibroblasts appear to be a sensitive assay for carcinogenic and mutagenic nitrosation products which may be formed within an organism from non-carcinogenic compounds.     

Inhibition of protein and DNA synthesis in tissue culture cells by a derivative of methyl glyoxal and ascorbate

The inhibitory effect of a methyl glyoxal-ascorbate (MGA) adduct (NFCR 278021) on protein and DNA synthesis in monolayer cultures of GPK epithelial cells has been compared with the inhibitory action of methyl glyoxal (MG). GPK cells exhibited an ID₅₀ of 0.98 μM MG for both protein and DNA synthesis compared with an ID₅₀ of 0.92 mM for the adduct. Hill plots demonstrate that the characteristics of the receptor saturation are the same for MG and MGA, suggesting that the action of the two agents is mediated through the MG moiety which is modified by the presence of the ascorbate portion of the molecule in MGA. It is shown that MGA undergoes spontaneous oxidation in solution and is a substrate for ascorbate oxidase, but that no additional MG activity is released by total enzymic oxidation of MGA, and oxidised MGA possesses the same inhibitory characteristics as MGA. Inhibition of protein synthesis by ascorbate or dehydroascorbate were not demonstrated in the dose range employed for MGA. The inhibitory effect of the adduct on protein synthesis was found to be diminished in the presence of glutathione and glyoxalase I (Glo I) and II (Glo II).     

Measurement of genotoxic activity in multiple tissues following inhalation exposure to dimethylnitrosamine

Chemically-induced DNA repair was measured as unscheduled DNA synthesis (UDS) in selected tissues isolated from rats following in vivo exposure to inhaled dimethylnitrosamine (DMN). UDS was evaluated in epithelial cells from rat nasal turbinates and trachea, in hepatocytes and in pachytene spermatocytes from the same treated animal. At nominal concentrations of 500 and 1000 ppm of DMN in air, chemically-induced DNA repair was observed in the epithelial cells of the upper respiratory system. DMN also entered the circulation, as evidenced by a strong DNA-repair response in hepatocytes. No DNA repair was observed in pachytene spermatocytes indicating either that DMN or its active metabolites did not reach the testes in sufficient concentration to induce DNA repair or that the testes lacked the capability to metabolically activate the compound. These results illustrate the potential of this approach to assess the organ-specific genotoxicity of environmental chemicals.     

The inhibitory effect of whole and deproteinized saliva on mutagenicity and clastogenicity resulting from a model nitrosation reaction

The objective of this study was to simulate in vitro at least some of the conditions that prevail in man during ingestion of nitrate and nitrosable compounds. Human saliva has been chosen because most chemicals ingested through food will interact with saliva. The nitrosation of methylurea was used as a model because the nitrosation products can be readily detected by their mutagenic (his+ revertants of S. typhimurium) and clastogenic (chromosome aberrations in CHO cells) properties. The results show that human saliva inhibits the formation of mutagenic and clastogenic nitrosation products when present during nitrosation. A 50% inhibition of mutagenicity results from the addition of a saliva sample diluted at 5% of the original concentration. In the test system used a similar inhibitory effect was obtained by 2.5 mM ascorbic acid or 2.0 mM chlorogenic acid. The main inhibitory agents seem to reside in a deproteinized fraction which was filtered through an ultrafilter UM2 (greater than 1000 MW). At strong acid levels (below pH 2) the saliva loses its inhibitory effect on the nitrosation of methylurea. The contribution of saliva to the inhibition of endogenous nitrosation within the oral cavity or stomach is discussed.     

Mutagenicity of Maillard browning reaction products from various nitrosated amino acid-glucose mixtures

Ten different amino acid-glucose Maillard browning products before and after reaction with nitrite were evaluated by the Ames mutagenicity assay. No mutagenic response was observed in the methylene chloride extracts of any browning products tested before nitrosation. However, mutagenicity was showed in most of the browning mixtures, e.g., glycine-glucose, lysine-glucose (I), arginine-glucose, phenylalanine-glucose (II), and methionine-glucose after nitrosation when examined by Salmonella typhimurium strains TA98 and TA100 either with or without S-9 metabolic activation. Among the browning mixtures, (I) and (II) showed the greatest mutagenic activity after reaction with nitrite. The mutagenicity of lysine-glucose with nitrite was dependent on browning intensity, nitrosation pH, nitrosation time, nitrite level and blocking agents.     

Sensitization of rad mutants of Dictyostelium discoideum to ultraviolet light by postirradiation treatment with caffeine

The capacity of normal human cells to regulate DNA-repair pathways was examined. Synchronous populations of WI-38 human diploid fibroblasts were used to determine whether base-excision repair was increased as a function of the cell cycle. 2 parameters of the base-excision repair pathway were examined: (1) The induction of the DNA-repair enzyme uracil DNA glycosylase which functions in an initial step in base excision repair: (2) cell-mediated base-excision repair as measured by unscheduled DNA synthesis after exposure to sodium bisulfite or to methyl methanesulfonate. The glycosylase activity was increased 5-fold during cell proliferation; unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fashion. Equivalent results were observed where repair replication was quantitated using density-gradient analysis in the absence of hydroxyurea. The increase of the activity of the uracil DNA glycosylase and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication both glycosylase activity and DNA repair had substantially diminished. As the cells entered the second cell cycle, the glycosylase activity was again increased and then was again diminished. These results suggest that human cells actively modulate this DNA-repair pathway. The temporal stimulation of base-excision repair suggests the possibility that a DNA-repair complex may be formed prior to DNA replication to prescreen DNA and thus ensure the transfer of the correct genetic information to daughter cells.     

Corticosteroids inhibit the delivery of short-term activational pulses of phorbol ester and calcium ionophore to human peripheral T cells.

Although there is evidence that corticosteroids inhibit receptor-ligand-induced phospholipid hydrolysis, the immunosuppressive effects of these agents downstream of protein kinase C (PK-C) activation and cytosolic Ca2+ mobilization is unclear. Previous studies indicated that T cell proliferative activation could be achieved with simultaneous short-term (e.g., 15-120 min) exposure to agents activating PK-C and elevating cytosolic Ca2+. In the studies reported here, similar procedures were utilized for determining whether corticosteroids alter T cell activation signals downstream of second messenger events. Dexamethasone interfered with T cell activation induced by short-term exposure to phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore, ionomycin. The inhibitory effect was evident with as little as 15 min of exposure to dexamethasone and T cell activating agents, making mechanisms involving de novo protein synthesis unlikely. Dexamethasone's effects in this system were blocked by the steroid receptor antagonist RU-486, indicating that the inhibition was mediated through the glucocorticoid receptor. The inclusion of recombinant interleukin-2 (IL-2) only partially overcame the dexamethasone inhibitory effect. Long-term (i.e., 48 hr) direct stimulation of PK-C with either PDBu or the non-tumor-promoting PK-C activator, bryostatin 1, also substantially overcame dexamethasone's effects, resulting in a recovery of IL-2 production and significant restoration of the T-cell proliferative response. These observations suggest that treatment with a PK-C-activating agent such as bryostatin 1 could reduce glucocorticosteroid-induced immunosuppression.     

Tissue-specific inhibition of [3H]thymidine incorporation into DNA by carcinogenic N-nitrosamines

The N-nitrosamines N-nitrosodimethylamine (DMN), N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were injected intraperitoneally 24 h before sacrifice in F344 rats and C57BL mice in doses of 297 mumoles/kg b.w. and 148 mumoles/kg b.w., respectively. 2 h before sacrifice, the animals were given an intraperitoneal injection of [3H]thymidine. The results showed that the examined N-nitrosamines inhibited the incorporation of [3H]thymidine into DNA in a few tissues of the rats and the mice. The results indicated that the N-nitrosamines exerted a tissue-specific inhibition of the [3H]thymidine incorporation in the tissues reported to be involved in the biotransformation of these substances. The observed inhibitory effects on the incorporation of [3H]thymidine by DMN, NNN and NNK were also correlated to a considerable extent to the reported sites of carcinogenicity. The present study indicates that measurements of [3H]thymidine incorporation into DNA in various tissues of experimental animals is a useful short-term bioassay to evaluate the potential tissue-specific carcinogenicity of the N-nitrosamines. The method may also be useful as a complement to other short-term in vivo tests in the screening of potential genotoxicity of several other chemicals.     

Conversion of arachidonic acid to prostanoids in the avian uterus

In an effort to obtain information on the possible source of prostaglandins which have been shown to play an important role in oviposition we examined the metabolism of arachidonic acid in microsomal preparations of both the muscular and the glandular tissue of the hen uterus. We found that adrenaline and tryptophan (but not hydroquinone) were effective stimulators of prostanoid synthesis. On incubation with [3H]arachidonic acid we identified, using TLC radiochromatography and several solvent systems, prostaglandins F2 alpha and E2 and, predominantly, thromboxane B2 which could not be attributed to platelet contamination. Addition of reduced glutathione increased prostaglandin E2 formation at the expense of thromboxane B2 and at 1 mM concentration suppressed adrenaline-promoted prostanoid synthesis. While the former effect has been documented in many other systems and could be ascribed to the activation of prostaglandin H2 to prostaglandin E2 isomerase, the latter effect is postulated to be due to an inhibition of cyclooxygenase. Interestingly, this inhibitory effect was shared by a number of reducing agents. Although the subcellular preparations were derived from structurally and functionally different tissues, there was no qualitative difference with respect to prostanoid synthesis. Our data support the role of locally produced primary prostaglandins in the regulation of oviposition and raise the question of a potential role for thromboxane in this process.     

Effects of human interferon-alpha on UV-induced DNA-repair synthesis and cell killing

Cells of a human transformed cell line, RSb, which have unusually high sensitivity to UV killing and low DNA-repair capacity and which can be induced into antiviral states by human interferon (HuIFN)-alpha (Suzuki et al., 1982), acquired increased levels of DNA-repair synthesis and cell survival when pretreated with HuIFN-alpha before UV irradiation. The increased effects of HuIFN-alpha were also observed in other transformed VA13 cells and AK fibroblast cells.     

The Frontal Eye Fields Limit the Capacity of Visual Short-Term Memory in Rhesus Monkeys

The frontal eye fields (FEF) in rhesus monkeys have been implicated in visual short-term memory (VSTM) as well as control of visual attention. Here we examined the importance of the area in the VSTM capacity and the relationship between VSTM and attention, using the chemical inactivation technique and multi-target saccade tasks with or without the need of target-location memory. During FEF inactivation, serial saccades to targets defined by color contrast were unaffected, but saccades relying on short-term memory were impaired when the target count was at the capacity limit of VSTM. The memory impairment was specific to the FEF-coded retinotopic locations, and subject to competition among targets distributed across visual fields. These results together suggest that the FEF plays a crucial role during the entry of information into VSTM, by enabling attention deployment on targets to be remembered. In this view, the memory capacity results from the limited availability of attentional resources provided by FEF: The FEF can concurrently maintain only a limited number of activations to register the targets into memory. When lesions render part of the area unavailable for activation, the number would decrease, further reducing the capacity of VSTM.     

Genotoxic and carcinogenic risk to humans of drug-nitrite interaction products

The large majority of N-nitroso compounds (NOC) have been found to produce genotoxic effects and to cause tumor development in laboratory animals; four NOC have been classified by the International Agency for Research on Cancer (IARC) as probably and another 15 as possibly carcinogenic to humans. A considerable fraction of drugs are theoretically nitrosatable due to the presence of amine, amide or other groups which by reacting with nitrite in the gastric environment, or even in other sites, can give rise to the formation of NOC, and in some cases other reactive species. This review provides a synthesis of information on the chemistry of NOC formation, the carcinogenic activity of NOC in animals and humans and the inhibitors of nitrosation reactions. It contains information on the drugs which have been tested for the formation of NOC by reaction with nitrite and the genotoxic-carcinogenic effects of their nitrosation products. In an extensive search we have found that 182 drugs, representing a wide variety of chemical structures and therapeutic activities, were examined in various experimental conditions for their ability to react with nitrite, and 173 (95%) of them were found to form NOC or other reactive species. Moreover, 136 drugs were examined in short-term genotoxicity tests and/or in long-term carcinogenesis assays, either in combination with nitrite or using their nitrosation product, in order to establish whether they produce genotoxic and carcinogenic effects; 112 (82.4%) of them have been found to give at least one positive response. The problem of endogenous drug nitrosation is largely unrecognized. Only a small fraction of theoretically nitrosatable drugs have been examined for the possible formation of genotoxic-carcinogenic NOC, guidelines for genotoxicity testing of pharmaceuticals do not indicate the need of performing the appropriate tests, and patients are not informed that the drug-nitrite interaction and the consequent risk can be reduced to a large extent by consuming the nitrosatable drug with ascorbic acid.     

DNA fragmentation by N-nitrosodimethylamine and methyl methanesulfonate in human hepatocyte primary cultures

The ability of N-nitrosodimethylamine (DMN) and methyl methanesulfonate (MMS) to induce DNA damage in primary cultures of human hepatocytes was examined by the alkaline elution technique. Both the agents induced a dose-dependent increase in DNA elution rate, but appreciable differences in the degree of response to the procarcinogen DMN were observed among cultures obtained from the livers of four patients. A comparative analysis of DNA fragmentation indicated a substantial similarity between human and concurrently studied rat hepatocytes in their response to both DMN and MMS.     

Age-related variation in the DNA-repair synthesis after UV-C irradiation in unstimulated lymphocytes of healthy blood donors

UV-C light-induced DNA-repair synthesis was studied in unstimulated lymphocytes of 51 healthy blood donors aged between 17 and 74 years. Repair synthesis was measured by the incorporation of [³H]thymidine after a 2-h incubation period in the presence of 2 mM hydroxyurea. The evaluation included (1)the spontaneous DNA-synthesis in unirradiated lymphocytes with and without hydroxyurea, (2) the DNA-repair synthesis in lymphocytes irradiated with UV-light.The interindividual variation was significantly higher than the methodological variation ascertained in 24 persons in whom 2 determinations were carried out.In blood donors aged between 17 and 39 years, the spontaneous DNA synthesis, both with and without hydroxyurea, was significantly lower than in older individualsThe DNA-repair synthesis was dependent on the dose of UV-C light between 2 and 16 J/M². There were no significant differences in DNA-repair synthesis in the age range 17–74 years. The variations in rate of DNA-repair synthesis (greater or lesser degree of [³H]thymidine incorporation beyond the 99% confidence range) were wider in older (44–74 years), than in younger individuals.     

The effect of paracetamol on oxidative damage in human peripheral lymphocytes

Unscheduled DNA synthesis (UDSox) and lipid peroxidation (LPO) induced by non-enzymatic activation of molecular oxygen (Fe2+ +H2O2) were measured in human peripheral lymphocytes from healthy volunteers. The effect of paracetamol (PC) in a final concentration range of 0.05-10 mmole/l on these oxidative processes and on DNA repair induced by MNNG (UDSmut) was investigated. The level of induced LPO was measured by the thiobarbituric acid assay, UDSox and UDSmut were determined by scintillometric measurement of incorporated [methyl-3H]thymidine into damaged DNA. PC at concentrations lower than 1 mmole/l significantly potentiates the non-enzymatically induced LPO and UDSox with the maximum of the activation being around 0.1 mmole/l. In contrast, PC at concentrations higher than 1 mmole/l exhibits an inhibitory effect on both LPO and UDSox. On the other hand, concentrations higher than 1 mmole/l significantly suppressed DNA-repair synthesis induced by MNNG.     

Cytotoxicity and SOS-inducing ability of ethidium and photoactivable analogs on E. coli ethidium-bromide-sensitive (Ebs) strains

In a recently-characterized ethidium-bromide-sensitive E. coli strain, DNA appears to be much more accessible to DNA-binding agents. This strain therefore appears to be of interest for studying the mutagenic properties of chemicals. For this purpose, a series of ethidium-sensitive E. coli strains (Ebs) with normal and defective DNA-repair capacity was constructed and made lysogenic for lambda (sfiA::lacZ). These strains were used to study the cytotoxicity and SOS-inducing ability of ethidium and its two photoactivable analogs 8-azido- and 3,8-diazido-ethidium. When non-covalent DNA complexes are formed, these dyes elicit only a bacteriostatic effect in the Ebs strains, which is almost independent of the strain's DNA-repair capacity. The SOS system is not induced. When covalent DNA adducts are formed after photoactivation of ethidium azido analogs, the effects are quite different. The formation of about 5 DNA monoadducts per cell induces a lethal hit in the Ebs uvrB recA strain and measurable SOS induction in the Ebs uvrB (lambda (sfiA::lacZ) strain. The formation of more than 1000 DNA adducts in the Ebs strain with normal DNA-repair capacity does not induce any measurable cytotoxic effect.     

Increased Intrinsic Connectivity of the Default Mode Network in Temporal Lobe Epilepsy: Evidence from Resting-State MEG Recordings

The electrophysiological signature of resting state oscillatory functional connectivity within the default mode network (DMN) during spike-free periods in temporal lobe epilepsy (TLE) remains unclear. Using magnetoencephalographic (MEG) recordings, this study investigated how the connectivity within the DMN was altered in TLE, and we examined the effect of lateralized TLE on functional connectivity. Sixteen medically intractable TLE patients and 22 controls participated in this study. Whole-scalp 306-channel MEG epochs without interictal spikes generated from both MEG and EEG data were analyzed using a minimum norm estimate (MNE) and source-based imaginary coherence analysis. With this processing, we obtained the cortical activation and functional connectivity within the DMN. The functional connectivity was increased between DMN and the right medial temporal (MT) region at the delta band and between DMN and the bilateral anterior cingulate cortex (ACC) regions at the theta band. The functional change was associated with the lateralization of TLE. The right TLE showed enhanced DMN connectivity with the right MT while the left TLE demonstrated increased DMN connectivity with the bilateral MT. There was no lateralization effect of TLE upon the DMN connectivity with ACC. These findings suggest that the resting-state functional connectivity within the DMN is reinforced in temporal lobe epilepsy during spike-free periods. Future studies are needed to examine if the altered functional connectivity can be used as a biomarker for treatment responses, cognitive dysfunction and prognosis in patients with TLE.