Net Grains Research Articles

Food Security in Kashmir: Food Production and the Universal Public Distribution System

India has been witnessing political conflicts in many parts of the country which have had severe implications for people living in these areas. In the context of Kashmir, which has seen conflict for more than two decades, this article investigates trends in local food production and access to food grains under the public distribution system (PDS). It also explores whether a programme based on the principle of universality can make a difference to outcomes against a backdrop of conflict and strife. Relying on an analysis of a combination of macro data and primary research, the article finds that the food deficit increased in the state post 1990s, temporal with the onset of conflict, but concludes that with net grains imported through PDS, the state has been, in effect, a food surplus state, which ensures that almost every household is entitled and has access to a food ration at a constant price.

The Design of Small Agricultural Corn Thresher

This design is based on the structure of various thresher, the main research of the corn thresher drive system structure, working principle, and on this basis, based on the design of the finished parts, on corn dish itself characteristic to carry on the design, when threshing grain rod roller high-speed rotation, the corn by continual lines between grid type and gravure roller rod, and the action between the corn cob constantly collide with each other, to achieve the seed plate separation, then the net grains, complete threshing.

CHARACTERISTICS OF A NEW JAPANESE PEAR CULTIVAR, 'FUKUSUI'

‘Fukusui’ is a new Japanese pear cultivar, selected from the seedlings of ‘Chikusui’ by Mr. Fujii, Tsu city, Mie prefecture. The fruit ripens during early to middle September at the original site, Hisai city. Anthesis is earlier, about the same as ‘Housui’ and ‘Choujuurou’. Low cross compatibility with ‘Kousui’ but high with other major cultivars. The amount of net pollen grains is 0.125g per 100 flowers which is less than that of ‘Chikusui’ and similar to that of ‘Housui’. Fruit is large and abundant, 467g in wt., skin color is reddish brown, redder than ‘Housui’ and ‘Choujuurou’. Fruit hardness is 5.4 lb, somewhat harder than ‘Housui’ but softer than ‘Choujuurou’. Brix is 12.7, higher than ‘Housui’, and it contains more fructose and so sweeter. pH is 4.65, similar to ‘Housui’ and somewhat tart. This cultivar is balanced with acidity and has fine texture, thus palatability is excellent. Bagging culture results in lowering of Brix by 0.5, poor development of cork tissue in dot, and spreading of thinner cork development between dots. It is resistant to many pests and diseases and there is few occurrence of spray injury. Growing of the new cv. ‘Fukusui’ will gradually increase as it matures early- to mid-September, in the zones where russet type cultivars of Japanese pear have been grown.

Lack of effect of coumarin on unscheduled DNA synthesis in the in vivo rat hepatocyte DNA repair assay

The ability of coumarin to induce UDS in male Sprague–Dawley CD rat hepatocytes in vivo was assessed using the unscheduled DNA synthesis (UDS) assay. From a preliminary toxicity study the oral maximum tolerated dose (MTD) of coumarin was determined to be 320 mg/kg body weight. For the UDS studies, rats were treated with 0 (corn oil control), 32 (one-tenth the MTD), 107 (one-third the MTD) and 320 (MTD) mg/kg coumarin via oral gavage. Rats were also treated with 20 mg/kg body weight dimethylnitrosamine (DMN) or 50 mg/kg body weight 2-acetylaminofluorene (2-AAF) as positive controls for the 2–4 hr and 12–16 hr expression of UDS, respectively. Hepatocytes were isolated by liver perfusion either 2–4 hr or 12–16 hr after treatment and cultured in medium containing [ methyl- 3H]thymidine for 4 hr and assessed for UDS by grain counting of autoradiographs. Coumarin treatment at doses of 32–320 mg/kg body weight had no statistically significant or dose-related effect on UDS in rat hepatocytes either 2–4 hr or 12–16 hr after dosing. In contrast, both DMN 2–4 hr after dosing and 2-AAF 12–16 hr after dosing produced significant increases in UDS assessed as the net nuclear grain count. Both genotoxins also increased the percentage of hepatocyte nuclei with greater than 5 net grains. Treatment with coumarin, DMN and 2-AAF had no statistically significant effect on the proportion of rat hepatocytes undergoing replicative DNA synthesis. In summary, this study demonstrates that coumarin does not induce UDS in hepatocytes of male Sprague–Dawley CD rats after oral administration at doses up to the MTD of 320 mg/kg. The responsiveness of the animals used in this study to genotoxic agents was demonstrated by the clear induction of DNA repair after treatment with DMN and 2-AAF.

Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo.

Pyridine was evaluated in an in vivo/in vitro mouse DNA repair assay. Unscheduled DNA synthesis (UDS) was used as an indicator of DNA damage to hepatocytes from male B6C3F1 mice. Test animals were exposed by oral gavage to pyridine or to the vehicle or positive control articles, and hepatocytes were collected and labeled by incubation in media supplemented with [3H]thymidine. Following labeling, the cultures were processed for autoradiographic analysis. Doses were selected based on a pilot study in which 0, 250, 500, 750, 1000 or 2000 mg kg(-1) pyridine in water was administered by gavage. Mice in the 1000 and 2000 mg kg(-1) dose groups were comatose following dosing and died within 24 h of dose administration. Pyridine dose levels for the UDS determination were set at 175, 350 and 700 mg kg(-1). Pyridine solutions in water were administered to mice 2 or 16 h prior to the scheduled sacrifice. The vehicle control group received water 16 h before sacrifice and the positive control group received 10 mg kg(-1) dimethylnitrosamine (DMN) 2 h before sacrifice. Pyridine did not significantly increase the UDS response in hepatocytes isolated from the treated animals, as measured by the incorporation of [3H]thymidine, using standard criteria for a negative response: less than zero mean net grains in repair (NG) and <20% of cells in repair (% IR; cells in repair have at least 5 NG). The vehicle control group and the low, mid- and high pyridine dose groups yielded less than -8.3 NG and < or =1% IR. The positive control group yielded a positive UDS response, with 10.8 NG and 62% IR. These results indicate that pyridine is non-genotoxic in B6C3F1 mouse liver using the UDS endpoint.

Lack of Effect of Coumarin on Unscheduled DNA Synthesis in Precision-cut Human Liver Slices

In this study the effect of coumarin on unscheduled DNA synthesis (UDS) in precision-cut human liver slices has been examined. Liver slices from tissue samples from four donors were cultured for 24 hr in medium containing [ 3H]thymidine and 0–5.0 m m coumarin using a dynamic organ culture system and processed for autoradiographic evaluation of UDS. As positive controls liver slices were also cultured with three known genotoxic agents, namely 0.02 and 0.05 m m 2-acetylaminofluorene (2-AAF), 0.002 and 0.02 m m aflatoxin B 1 (AFB 1) and 0.005 and 0.05 m m 2-amino-1-methyl-6-phenylimidazo [4,5- b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with more than five net grains. Compared with control liver slice cultures, treatment with 0.05–5.0 m m coumarin had no effect on UDS. In contrast, treatment with 0.02 and 0.05 m m 2-AAF, 0.002 and 0.02 m m AFB 1 and 0.005 and 0.05 m m PhIP produced significant increases in the net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 m m PhIP. Treatment with 2-AAF, AFB 1 and PhIP also produced significant increases in the number of centrilobular hepatocyte nuclei with more than five net grains. At the concentrations examined neither coumarin, 2-AAF, AFB 1 nor PhIP had any significant effect on replicative DNA synthesis in 24 hr cultured human liver slices. These results demonstrate that coumarin does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice preparations used in this study. The results of this study suggest that coumarin is not a genotoxic agent in human liver.

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7 H-pyrrolo[2,3- d]pyrimidine (U-89843)

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1–40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.

Supervising skill information and violation of environmental regulations

Conditions under which an enforcement agency gains from disseminating information on its skill on detecting and convicting violators are analyzed. This seems to be a very attractive enforcement strategy because it can be accomplished at no or a very low cost. It is assumed that the enforcement agency signals when this implies net grains, which include losses from violation, such as negative environmental benefits and charge payments, and benefits from violation, i.e., expected fine payments of detected violations. The analytical results show that the impact on violation and associated net gains depends on (i) information strategies applied by the skillful and less skillful enforcement agency, respectively, and (ii) the relation between losses and expected fine payments for violations. When the information strategy implies a relatively high (low) probability for a skillful enforcement agency to reveal information, dissemination of information generates net gains when the losses from violation exceed (are below) the expected fine incomes. An application to regulation on farmers' manure treatment practices in Gotland shows that signaling may reduce or increase violation by about 25% depending on the signaling strategies. The calculated net benefits, the difference in environmental benefits, and expected fine payments, are, however, small in magnitude under all signaling strategies.

Lack of effect of piperonyl butoxide on unscheduled DNA synthesis in precision-cut human liver slices

In this study the effect of piperonyl butoxide (PBO) on unscheduled DNA synthesis in precision-cut human liver slices has been examined. Liver slices prepared from tissue samples from five human donors were cultured in medium containing [ 3H]thymidine and 0–2.5 mM PBO using a dynamic organ culture system. After 24 h the liver slices were processed for autoradiographic examination of UDS. As positive controls, liver slices were also cultured with three known genotoxic agents, namely 2-acetylaminofluorene (2-AAF), aflatoxin B 1 (AFB 1) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with >5 and >10 net grains. Compared to control liver slice cultures PBO had no effect on UDS. In contrast, treatment with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 and 0.02 mM AFB 1 and 0.005 and 0.05 mM PhIP produced significant increases in net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 mM PhIP. Treatment with 2-AAF, AFB 1 and PhIP also produced increases in the number of centrilobular hepatocyte nuclei with >5 and >10 net grains. At the concentrations examined neither PBO, 2-AAF nor PhIP had any significant effect on replicative DNA synthesis in 24 h cultured human liver slices. In cultured liver slices treated with 0.02, but not 0.002, mM AFB 1 a significant reduction in the rate of replicative DNA synthesis was observed. These results demonstrate that PBO does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice

Definition of a new intralaboratory limit value for a positive UDS assay in vitro

In our laboratory, we routinely perform the unscheduled DNA synthesis (UDS) assay in vitro using rat primary liver cells and autoradiographical evaluation. The fixed limit of +5 nuclear net grains (NNG) for a positive UDS response no longer seems to be appropriate because of the advanced techniques that are now used. Considering our historical data (mean vehicle control NNG of 45 experiments = −1.5; highest observed mean NNG value in 126 control slides = +1.63 NNG) we will use a mean value of +2 NNG as our new intralaboratory limit value for a positive UDS assay, independent of the statistical evaluation. Experiments with a mean NNG count in the vehicle control of > +0.5 will be rejected as invalid and repeated since mean values of > +0.23 NNG were never reached. Additionally, the NNG data obtained will be subjected to a newly proposed statistical method which includes both the actual and the mean historical control NNG value. First, the sum of the actual and the mean historical control value is divided by two. Secondly, the calculated control value is pairwise compared with the treated groups by the one-sided Dunnett's test. This might be a helpful tool to evaluate a weak or equivocal UDS response.

Effects of sterigmatocystin and T-2 toxin on the induction of unscheduled DNA synthesis in primary cultures of human gastric epithelial cells.

Primary cultures of human gastric epithelial cells were tested for induction of unscheduled DNA synthesis (UDS) by sterigmatocystin (ST) and T-2 toxin. Autoradiographic results indicated that ST (10(-6)-10(-4)M) induced UDS in the presence of S9 activation system. The repair rates were 24-91% (net grains > or = 3) and 2-71% (net grains > or = 5). T-2 toxin did not induce UDS in this study.

Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes

Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene [B(a)P], (+/-)7 beta-8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)anti-BPDE], and (+/-)7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)syn-BPDE] to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present in the cell population when comparing similar compounds within the linear dose-response range of 0.005 micrograms/ml-0.25 micrograms/ml. A higher rate of UDS for a given level of DNA adducts was interpreted as an increased efficiency of DNA repair. In some cases, an increase in the efficiency of DNA excision repair correlated with lower tumor-initiating activity. For this family of PAH, the concentration below which UDS could no longer be detected was approximately 0.01 microgram/ml. However, DNA adducts were measurable at concentrations of 0.01 and 0.005 micrograms/ml. The limits of detection of the current UDS assay in the SENCAR MEK culture system occurred at hydrocarbon adduct levels of approximately 10 pmol/mg DNA, or approximately 1 adduct per 3 x 10(5) bases. Additionally, the UDS assay was unable to detect DNA repair induced by the weakly carcinogenic PAHs, dibenz(aj)anthracene and 7-methyl-dibenz(aj)anthracene. The UDS assay did detect DNA repair by the more strongly carcinogenic PAH, 6-methylcholanthrene. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.

Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres

Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G 0/G 1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [ 3H]thymidine incorporation([ 3H]dThd), RPMCs treated with 4 μg/cm 2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microsopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [ 3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [ 3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 μg/ml of benzo[ a]pyrene resulted in a significant increase in[ 3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [ 3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibers in the absence of HU resulted in a reduction of [ 3H]dThd incorporation attributed to an impairment of the S phase by the fibers. 1–4 μg/ml benzo[ a]pyrene or 10–50 J/m 2 UV light resulted in an approximate doubling of [ 3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [ 3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS and RPMCs.

The presence of leukotriene C4-binding sites in bovine corpora lutea of pregnancy.

The presence of binding sites for [3H]leukotriene (LT) C4 in bovine corpora lutea of pregnancy was investigated with quantitative light microscopic autoradiography. Silver grains were found over small (15-20 microns) and large (20-50 microns) luteal cells and arteriolar smooth muscle. Vascular endothelial cells, erythrocytes in arteriolar lumen, and fibroblasts, on the other hand, contained very few or no net grains. The grain distribution over luteal cells and arteriolar smooth muscle was reduced (p less than 0.001) after coincubation with excess unlabeled LTC4 but not with excess unlabeled LTA4, LTB4, LTD4, LTE4, prostaglandin (PG)E2, PGF2 alpha or PGI2. The large luteal cells contained 16.1 net grains per cell, which was 6.4 and 7.0 times the number of specific grains as in small luteal and arteriolar smooth muscle cells, respectively (p less than 0.001). When the net grains were corrected for cell area differences, large luteal cells and arteriole smooth muscle cells contained a similar number of grains-which was two times as many as those found in small luteal cells. These findings suggest that LTC4 can potentially regulate functions of not only luteal cells but also luteal vasculature.

Measurement of unscheduled DNA synthesis in primary cultures of adult mouse epidermal keratinocytes.

Skin is a major target tissue for many environmental carcinogens. In order to provide a tool to study the role of DNA-repair processes in relation to DNA damage and carcinogenesis in this tissue, we have developed an assay that measures chemically induced DNA repair as unscheduled DNA synthesis (UDS) using cultures of adult mouse epidermal keratinocytes (MEKs) from SENCAR mice. Primary MEKs were prepared and incubated for 24 h in the presence of the test chemical and 10 microCi/ml [3H]thymidine. UDS was quantitated autoradiographically as net grains per nucleus. This assay detected DNA damage caused by the direct-acting agents N-methyl-N'-nitro-N-nitrosoguanidine, N-methylnitrosourea, methyl methanesulfonate, ethyl methanesulfonate and (+/-)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene and the indirect-acting carcinogens 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, adriamycin and 4-nitroquinoline-1-oxide. The growth conditions used allowed the epidermal cells to retain the tissue specificity of carcinogen activation of mouse epidermis in vivo in that 2-acetylaminofluorene was inactive in this assay. This assay system should be useful for determining genotoxic and potential carcinogenic agents for skin as well as for mechanistic studies involving DNA repair and chemical carcinogenesis in this tissue.

Background DNA repair synthesis in rat hepatocyte cultures used for genotoxicity testing

DNA repair synthesis in primary rat hepatocyte cultures (HPC) was investigated using the bromodeoxyuridine (BrdUrd) density-shift method and autoradiography. Analysis by density-gradient centrifugation of DNA labelled with BrdUrd and [ 3H]deoxycytidine ([ 3H]dCyd) for 18–20 hr showed that considerable DNA repair replication occurs in HPC even in the absence of an added genotoxic agent. Repair was demonstrated to be most probably a consequence of DNA damage caused by the collagenase perfusion of the liver during hepatocyte isolation, and by β-decay of the 3H-labelled DNA precursor. Autoradiographic analysis of the distribution of silver grains over nuclei in intact non-S-phase cells, and over isolated nuclei from HPC exposed to [ 3H]dCyd for 18–20 hr, showed that the vast majority of the radioactivity was incorporated into the nuclei themselves, and not into overlying cytoplasm or mitochondria. In addition, direct localization of mitochondria in hepatocytes using the mitochondrion-specific dye rhodamine 123 showed that very few mitochondria were actually located over the nucleus. The results suggest that cytosolic labelling of control HPC in autoradiographs is mainly caused by mitochondrial DNA synthesis whereas nuclear labelling essentially reflects repair synthesis. They call into question the commonly applied practice of evaluating unscheduled DNA synthesis (UDS) in HPC by subtracting the number of cytosolic silver grains from nuclear grains and expressing repair synthesis as net grains per nucleus.

Evaluation of purified 4-deoxynivalenol (vomitoxin) for unscheduled DNA synthesis in the primary rat hepatocyte-DNA repair assay

Primary cultures of rat hepatocytes were used to determine unscheduled DNA synthesis (UDS) and cytotoxicity of purified 4-deoxynivalenol (vomitoxin), a trichothecene mycotoxin produced on cereal grains by fungi of the genus Fusarium. Nontoxic and toxic doses of deoxynivalenol, 0.1 to 1000 μg/ml, did not significantly increase UDS as measured by net grains per nucleus, net grains per nuclear area or percentage of cells incorporating ⩾ 5, 6, 10 or 20 grains per nucleus. Evidence of cytotoxicity, manifested as a reduction in cell number in autoradiographs, pyknotic nuclei or vacuolated cytoplasm, was observed in hepatocytes treated with deoxynivalenol concentrations of 5 μg/ml and above. These findings suggest that the cellular toxicity of deoxynivalenol may not be mediated by a DNA-damaging event in cultured hepatocytes. An increased percentage of large-sized nuclei was also found to be associated with toxic doses of deoxynivalenol as well as 2-acetylaminofluorene used as the positive control.

DNA repair synthesis induced by N-hydroxyurea, acetohydroxamic acid, and N-hydroxyurethane in primary rat hepatocyte cultures: comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increase over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genetoxicity may be a common property of aliphatic hydroxamic acids.

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes, Direct Blue 1 (DB1), Direct Blue 14 (DB14), Direct Brown 95 (DB95), and Direct Red 46 (DR46) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [ 3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [ 3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 μg/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.

Detection of unscheduled DNA synthesis in hepatocytes isolated from rats treated with genotoxic agents: an in vivo — in vitro assay for potential carcinogens and mutagens

An assay is described for the measurement of chemically-induced DNA repair in cultures of primary rat hepatocytes following in vivo treatment with genotoxic agents. Rats were exposed to chemicals then primary hepatocytes were isolated by liver perfusion and cultured with [3H]-thymidine. DNA repair was measured as unscheduled DNA synthesis (UDS) by quantitative autoradiography. Cells from control animals consistently ranged from -2 to -6 net grains (NG). Treatment of rats with 10, 1 or 0.1 mg/kg dimethylnitrosamine (DMN), i.p., yielded 36.6, 6.4 and -0.9 NG, respectively; 10 mg/kg DMN, per os (p.o.), produced 22.2 NG. Oral doses of 50 or 5 mg/kg acetylaminofluorene (AAF) yielded 14.0 and 6.4 NG, respectively. Carbon tetrachloride (CCl4) at 100 or 10 mg/kg, p.o., yielded -3.2 and -5.1 NG, respectively. Thus, dose-related increases in UDS were observed for the hepatocarcinogens DMN and AAF while vehicle controls and the hepatotoxin CCl4 produced no response. An examination of the time-course of DNA repair following DMN treatment shows a linear decline in UDS during the first 24 h post-treatment followed by a slower decline from 24 to 48 h. These results indicate that this assay is a potentially useful system for assessing the genotoxic and potential carcinogenic activity of chemicals in the whole animal.