Network on veterinary medicines initiated by the European Federation For Pharmaceutical Sciences.

The European Federation for Pharmaceutical Sciences (EUFEPS) was founded 25 years ago by more than 20 national pharmaceutical societies and faculty members. As a pan-European organization, it brings together pharmaceutical societies as well as academic, industrial and regulatory scientists engaged in drug research and development, drug regulation and education of professionals working in these fields. EUFEPS represents pharmaceutical sciences in Europe and is recognized as such by both the European Commission and the European Medicines Agency. EUFEPS cooperates with the European Federation of Pharmaceutical Industries and other European organizations and maintains global connections with agencies such as the US Food and Drug Administration and the American Association of Pharmaceutical Scientists. EUFEPS has established specified networks forming the basis of its activities. The creation of a Network on Veterinary Medicines is prompted by the manifold problems resulting from the use of veterinary drugs and its inherent interconnections with human medicine, environmental and public health. A long-term goal of this initiative was to expand the spectrum of available therapeutics for use in animals, including the development of innovative delivery systems.

Development and evaluation of a system to assess antimicrobial drug use in farm animals: results of an Austrian study.

The objective of this study was to develop and evaluate a feasible system for the collection of antimicrobial consumption data in farm animals in Austria. An electronic registry of all antibacterial pharmaceuticals approved in Austria for use in farm animals was created, listing product name, marketing authorisation number, active ingredient, package unit, strength, target species (cattle, swine, poultry), route of administration and indication, and allocating the corresponding code of the World Health Organization (WHO) Anatomical Therapeutic Chemical classification system for veterinary medicines to each substance (ATCvet-code). Different units (absolute quantities, animal daily dose, assumed daily product dose) enabled computation of the amounts of antimicrobials as pure substance, the constituents of a veterinary medicinal product, or the number of administrations. Two data collection systems were evaluated: (1) data transfer from the management software of veterinary practices or the Austrian Poultry Health Service; and (2) on-site data collection by manual data input from prescription records into an electronic registry. A total of 14,267 data sets provided by 18 practices were documented during the period January 2008 to March 2010. The total weight of active substances reported amounted to more than 5.4 tonnes for all species studied. The systems proved suitable for routine data acquisition and were considered in a recent national regulation on the surveillance of sale and consumption of veterinary antimicrobial substances.

The legal foundation of the production and use of herd-specific vaccines in Europe.

In veterinary medicine, herd-specific vaccines are primarily used in farm animals if traditional vaccines are either unavailable or do not perform as expected. As autogenous products, these vaccines are exempt from Directive 2001/82/EC, and therefore the production and use of herd-specific vaccines are regulated differently in each member state of the European Union (EU). This study is an overview of the diverse legal statuses of herd-specific vaccines among European countries. The study was conducted by analyzing legal documents, tailored questionnaires answered by subject-related authorities from sixteen European countries, and related literature. These analyses revealed that tremendous heterogeneity exists with respect to the legal requirements for the production and use of herd-specific vaccines among the countries that were examined. In particular, certain countries have detailed and precise regulations for these vaccines, whereas the legislation regarding these vaccines is vague or even nonexistent in other nations. The implementation of standardized definitions, guidelines for vaccine use in the field, and regulations for vaccine production are essential prerequisites for achieving legal consistency across Europe. These measures would also help countries enact pertinent national legislation with less divergence regarding the production and use of herd-specific vaccines and ensure the existence of comparable safety and quality standards for these vaccines among European countries.

Spontaneous oxidative DNA damage in bovine retained and nonretained placental membranes.

Retention of fetal membranes (RFM) is believed to be associated with conditions of oxidative stress. In this study, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was used for the determination of spontaneous oxidative DNA lesions in maternal and fetal parts of bovine retained and nonretained placentas. Placental specimens were collected directly after spontaneous delivery or during cesarean section from cows divided into 6 groups: (A) cesarean section before term without RFM, (B) with RFM, (C) cesarean section at term without RFM, (D) with RFM, (E) spontaneous delivery at term without RFM and (F) with RFM. Isolated DNA was hydrolyzed and analyzed by HPLC; native nucleosides were monitored at 254 nm and 8-OH-dG by electrochemical detection. No significant differences in 8-OH-dG levels between retained and nonretained placental tissues were found in all samples from preterm groups (mean concentrations between 13 and 42 micromol/mol deoxyguanosine (dG)). In the term cesarean section group with RFM a significant increase in 8-OH-dG concentration in DNA from maternal (8-fold) and fetal (18-fold) membranes were detected when compared to the respective nonretained tissues. Also, in the term spontaneous delivery groups maternal nonretained placental tissues showed increased levels of 8-OH-dG in comparison to the respective tissues of the retained placenta group. In placental tissues oxidative DNA lesions appear to be controlled by responsive mechanisms which, possibly following exhaustion, give rise to increased 8-OH-dG levels.

Levels of 8-hydroxy-2'-deoxyguanosine in cellular DNA from 12 tissues of young and old Sprague-Dawley rats.

The age dependent increase of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) reported in DNA of organs of old rats appears to vary with the strain, age and sex of the animals used for the investigation. Here we report on 8-OH-dG concentrations in the cellular DNA of 12 tissues of male Sprague-Dawley rats aged 5 or 30 months and kept under standard conditions throughout their lives until being killed. DNA from frozen organs was isolated using a Qiagen DNA purification kit. Following digestion (nuclease P1, alkaline nuclease) hydrolysed DNA was applied onto a HPLC column; native nucleosides were monitored at 254 nm and 8-OH-dG by electrochemical detection. 8-OH-dG levels in organs of young rats ranged between 10 and 90 micromol/mol deoxyguanosine (dG). Highest levels (micromol 8-OH-dG /mol dG) were detected in the oesophagus (90), muscle (61), brain (65), liver (59), spleen (57), and testicles (63). 8-OH-dG in DNA from kidney, lung, heart, small and large intestine ranged between 28 and 38 micromol/mol dG. Lowest amounts were found in the glandular stomach (10). DNA of old rats generally contained higher 8-OH-dG levels with significant increases in liver (186%) and kidney (372%); other organs showed no significant decreases (spleen, brain, testicles) or increases up to 69% (heart). These findings are discussed in the context with previously published data on 8-OH-dG levels in organs from young and old rats.

Analysis of polycyclic aromatic hydrocarbon-DNA adducts by postlabelling with the weak beta-emitters 35S-phosphorothioate and 33P-phosphate, immobilized boronate chromatography and high-performance liquid chromatography.

The wide range of carcinogenic activities of optical isomers of bay-region diol-epoxides of polycyclic aromatic hydrocarbons (PAHs) demands analytical techniques capable of distinguishing both the diastereoisomer and enantiomers of the PAH diol-epoxide (PAH-DE) responsible for DNA adduct formation. Our laboratory recently developed postlabelling procedures using the radioisotope 35S for HPLC analysis of benzo[a]pyrene (BaP)-DNA adducts formed in BaP-treated hamster cell cultures. To allow identification and complete separation of anti-PAH-DE-DNA adducts from syn-PAH-DE-DNA adducts, an immobilized boronate chromatography procedure was developed using a high-capacity boronate column. Analysis of 7,12-dimethyl-benz[a]anthracene (DMBA)-DNA adducts formed in treated hamster embryo cells by this boronate chromatography procedure and 35S-postlabelling demonstrated that both anti- and syn-DMBA-DE were bound to DNA. These analytical techniques also provided evidence that the potent carcinogen and environmental PAH dibenzo[a,l]pyrene was metabolized in cells to a DNA-binding intermediate with structural characteristics of an anti-PAH-DE. Conditions were also developed for using 33P-labelled ATP in the postlabelling assay. The use of [33P] instead of [35S]ATP offers the advantages of ease of labelling and greater sensitivity while still using a weak beta-emitter. The development of PAH-DNA adduct analysis techniques with improved sensitivity allows more detailed studies of how PAHs interact with DNA and how this leads to cancer induction.

High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.

Beta-deuteration of N-nitrosoethylmethylamine causes a shift in DNA methylation from rat liver to esophagus.

While N-nitrosoethylmethylamine (NEMA) is carcinogenic primarily for the liver, its beta-trideuterated derivative, N-nitroso( [2-D3]ethyl)methylamine (NEMA-d3), also produces a high incidence of tumors in the esophagus. To determine whether this shift in organ specificity is associated with an altered pattern of DNA alkylation, [methyl-14C]- and [1-ethyl-14C]-labeled NEMA-d3 were administered to adult male Fischer 344 rats as a single i.p. dose (0.05 mmol/kg; 4 h survival). Levels of methylated and ethylated purines in the DNA of various organs were determined by radio-chromatography on Sephasorb-HP columns. When compared to previous data using undeuterated NEMA, 7-methylguanine levels were found to be reduced by approximately 30% in liver and kidney, but were 160% greater in esophagus. This resulted in a decrease in the 7-methylguanine ratio for liver/esophagus from 109 to 29. O6-Methylguanine was diminished in liver and kidney, but levels in lung and esophagus were too low for quantitative detection. Similarly, deuteration led to an 18% decrease of 7-ethylguanine in hepatic DNA. The observed increase in esophageal DNA methylation correlates with the increased carcinogenicity of NEMA-d3 relative to undeuterated NEMA in that organ. Since pharmacokinetic studies have shown that beta-trideuteration of NEMA does not alter its bioavailability, the data suggest that the observed shift in target organ results from isotopically-induced changes in the balance among competing metabolic pathways in different rat tissues.

DNA methylation in rat stomach and duodenum following chronic exposure to N-methyl-N'-nitro-N-nitrosoguanidine and the effect of dietary taurocholate.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces a high incidence of carcinomas in the glandular stomach of rats following chronic administration in the drinking water. We determined the level of 7-methylguanine and O6-methylguanine in gastric and duodenal DNA during chronic exposure to MNNG (80 p.p.m.). After considerable fluctuations during the initial 3 weeks, levels of methylpurines reached a steady state which was approximately three times higher in the pylorus (i.e. the preferential site of tumor induction) than in the fundus and duodenum, with 7-methylguanine and O6-methylguanine values in the range of 520 and 110 mumol/mol guanine, respectively. When rats were given MNNG in the drinking water at concentrations ranging from 10 to 80 p.p.m. for 3 weeks, levels of methylpurines reached maximum values already at 10-20 p.p.m. At higher MNNG concentrations, there was no further increase in DNA alkylation. The reason for this lack of dose response remained unclear. Immunohistochemical analyses showed that DNA methylation by MNNG is restricted to epithelial cells bordering the luminal surface. The possibility exists that in this target cell population the content of free thiols is a limiting factor for the decomposition of MNNG and its reaction with macromolecules in the gastric mucosa. Addition to the diet of sodium taurocholate, a bile acid previously shown to enhance MNNG-induced stomach carcinogenesis, did not influence the extent of DNA methylation, indicating that it acts as a promoter.

Organ and cell specificity of DNA methylation by N-nitrosomethylamylamine in rats.

N-Nitrosomethylamylamine (NMAA) is a potent carcinogen in rodents with the esophagus as the principal target organ. The present study aims at an assessment of DNA methylation by NMAA in various rat tissues and an identification of cell populations actively involved in its bioactivation. Adult male F344 rats received a single i.p. dose of N-nitroso[methyl-14C]amylamine (0.1 mmol/kg). After 6 h organs were removed and the DNA was extracted, hydrolyzed in 0.1 M HCl, and subjected to radiochromatography on Sephasorb-HP. Highest levels of DNA alkylation were found in esophagus (798 mumol 7-methylguanine/mol mol guanine), followed by nasal epithelium (672 mumol) and liver (624 mumol). Trachea, lung, forestomach, and kidney had considerably lower levels of alkylation and in glandular stomach, spleen, and duodenum, values were close to the limit of detection. Specific target cell populations were identified autoradiographically and by immunohistochemistry using a rabbit antiserum to O6-methyldeoxyguanosine. In the esophagus, NMAA was selectively metabolized by the basal cells of the mucosa. In the respiratory tract, O6-methyldeoxyguanosine was almost exclusively present in the tracheal and bronchiolar epithelia. In the nasal cavity, labeled nuclei were found in both the olfactory and the respiratory epithelium and in the serous glands. Our studies indicate that NMAA and related asymmetrical nitrosamines are, in addition to liver, preferentially metabolized in tissues derived from the ventral entoderm, including the upper respiratory and gastrointestinal tract.

DNA methylation in rat tissues by a series of homologous aliphatic nitrosamines ranging from N-nitrosodimethylamine to N-nitrosomethyldodecylamine.

Aliphatic N-nitrosomethylalkylamines exhibit a remarkable organ specificity in rats, the principal targets for tumour induction being liver, oesophagus, urinary bladder and lung. We have determined the extent of DNA methylation in these tissues following a single oral dose (0.1 mmol/kg; 6 h survival) of each of 12 homologues, ranging from N-nitrosodimethylamine (C1) to N-nitrosomethyldodecylamine (C12). Methylpurines (7- and O6-methylguanine) were determined by cation exchange HPLC with fluorescence detection. Highest levels of hepatic DNA methylation were found with N-nitrosodimethylamine (C1) and N-nitrosomethylethylamine (C2), the most potent hepatocarcinogens in this series. Concentrations of methylpurines in liver DNA decreased with increasing chain length for C1-C5. Administration of the higher homologues (C6-C12) caused levels of DNA methylation which by themselves were considered too low to account for their hepatocarcinogenicity. In rat oesophagus, DNA methylation closely paralleled carcinogenicity, the butyl and pentyl derivatives (C4, C5) being most effective. In rat lung, the extent of DNA methylation was generally lower and there was no apparent correlation with carcinogenicity. Methylation of kidney DNA also decreased with increasing chain length and was only detectable for C1-C5. In urinary bladder DNA, methylpurines were below or close to the limit of detection. It is concluded that the initiation of malignant transformation by DNA methylation alone (through hydroxylation at the methylene alpha-carbon) could be operative for C1 in kidney and lung, for C1 and C2 in liver, and C3-C5 in oesophagus. For the higher homologues, the extent of DNA methylation seems insufficient to explain the complex pattern of tissue specificity, suggesting that DNA modification other than, or in addition to, methylation may be responsible.

Organ specificity, metabolism and reaction with DNA of aliphatic nitrosomethylalkylamines.

Aliphatic nitrosomethylalkylamines are carcinogens with a remarkable organ specificity in rats, the principal targets being liver, oesophagus and bladder. We have determined the extent of DNA methylation in these tissues following a single oral dose (0.1 mmol/kg; 6-h survival) of each of 12 homologues, ranging from N-nitrosodimethylamine (NDMA, C1) to N-nitrosomethyldodecylamine (C12). Methylpurines (7- and O6-methylguanine; 7-meGua and O6-meGua) were determined by cation-exchange high-performance liquid chromatography with fluorescence detection. Highest levels of hepatic DNA methylation were found with NDMA (C1) and N-nitrosoethylmethylamine (NEMA, C2), the most potent hepatocarcinogens in this series. Concentrations of methylpurines in liver DNA decreased with increasing chain length from C1 to C5. Administration of the higher homologues (C6-C12) caused levels of DNA methylation which by themselves were considered too low to account for their hepatocarcinogenicity. In rat oesophagus, DNA methylation closely paralleled carcinogenicity, the most effective agents being the butyl and pentyl derivatives (C4 and C5). Levels of DNA methylation in bladder epithelium were close to the limit of detection (C6,C9,C10,C12) and there was no apparent correlation with carcinogenicity. It is concluded that initiation of malignant transformation by DNA methylation alone (through hydroxylation of the nitrosamine at the methylene alpha-carbon) could be operative for C1-C5. For the higher homologues, this type of DNA modification is insufficient to explain the complex pattern of tissue specificity.

Induction of rat liver O6-alkylguanine-DNA alkyltransferase by bleomycin.

Alkyl adducts at the O6-position of guanine constitute promutagenic DNA lesions likely to be involved in the initiation of malignant transformation. They can be removed by a cellular acceptor protein termed O6-alkylguanine-DNA alkyltransferase (AT). In rat liver this repair enzyme can be induced by a variety of hepatotoxins, partial hepatectomy and X-irradiation. This paper describes a stimulation of the hepatic AT by treatment of rats with the radiomimetic agent, bleomycin. Induction of AT is dose-dependent up to 20 mg bleomycin/kg and appears to level off with higher doses. Enhancement of O6-meG repair is detectable within 24 h after a single i.p. injection. Maximum AT induction was reached after 6 days and amounted to 350% of the control levels. The enhancement of AT activity is not associated with acute liver injury and initially coincides with an inhibition of [3H]deoxythymidine incorporation into hepatic DNA. This indicates that AT induction in rat liver is not necessarily dependent on tissue necrosis with increased cell replication. Since bleomycin does not produce DNA lesions recognized and repaired by the AT, the hypothesis is entertained that AT induction by these agents is part of a concerted reaction to radiation-type DNA damage.

Metabolism of N-nitrosodimethylamine and N-nitrosomethylbenzylamine in domestic fowls: DNA alkylation and potential target sites for carcinogenicity.

The metabolism and reaction with DNA of N-nitrosodimethylamine (NDMA) and N-nitrosomethylbenzylamine (NMBzA) were investigated in chickens. These nitrosamines have been identified in a typical diet consumed in northern Chinese provinces with a high incidence of oesophageal cancer in both, humans and domestic fowls. Following i.m. or oral administration of the 14C-methyl labeled nitroso compounds (16.5 mumol/kg) less than 10% of the total radioactivity was exhaled as 14CO2 within 24 hr. DNA alkylation in chicken tissues was generally low, with highest values in liver (approximately 30 mumol N7-methylguanine/mol guanine) and kidney. In the gastrointestinal tract, concentrations of methylpurines were close to the limit of detection. After oral administration of NMBzA the reaction with DNA was more extensive, in particular in the diverticulum (crop), where N7-methylguanine and 0(6)-methylguanine amounted to 32 and 3.5 mumol/mol guanine, respectively. These values are more than 10-fold lower than those produced by a similar dose in rats, i.e. a species in which NMBzA induces a high incidence of oesophageal carcinomas. The low capacity of the chicken mucosa to bioactivate NDMA and NMBzA argues against these nitrosamines as agents responsible for oesophageal cancer in high risk areas.

Induction of rat liver O6-alkylguanine-DNA alkyltransferase following whole body X-irradiation.

The promutagenic DNA lesion O6-alkylguanine can be enzymically removed from cellular DNA by an O6-alkylguanine-DNA alkyltransferase (O6-AT) which transfers the alkyl group from the O6-position of guanine to a cysteine residue contained within its sequence. We report that whole body X-irradiation induces the O6-AT activity in the liver of adult Wistar rats. In vivo repair activity was assessed by radiochromatographic determination of the persistence of O6-methylguanine in hepatic DNA of irradiated rats following a single i.p. injection of N-nitroso-[14C]dimethylamine (2 mg/kg). In addition, the O6-alkylguanine repair capacity was assayed in vitro by incubation of extracts from whole liver, hepatocytes, or hepatic cell nuclei with 3H-methylated DNA and high performance liquid chromatography analysis of this DNA substrate. We found that whole body X-irradiation increased in a dose-dependent manner the O6-AT activity over a range of 100-800 R. The repair induction was first detectable 12 h after X-ray exposure, reached a maximum level at 72 h, and declined to control values within 12 days. Deoxythymidine incorporation into hepatic DNA was significantly reduced during the initial 72 h following application of 500 R. In a split dose irradiation schedule with daily doses of 15 R, a delayed enzyme induction occurred after a 30-day treatment period. Pretreatment of rats with the translational inhibitor cycloheximide completely suppressed the O6-AT stimulation. This indicates that the induction of hepatic O6-alkylguanine repair is due to de novo synthesis of alkyltransferase molecules.

Nafenopin-induced rat liver peroxisome proliferation reduces DNA methylation by N-nitrosodimethylamine in vivo.

The hypolipidaemic drug nafenopin (NAF) has been shown to enhance the hepatocarcinogenic effect of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine in rats. We have investigated whether the NAF-induced peroxisome proliferation in hepatocytes interferes with NDMA's metabolism and interaction with DNA. Adult male Wistar rats received a single i.p. injection of [14C]NDMA (2 mg/kg) and were killed 4 h later. DNA was isolated from liver and kidney, hydrolysed in 0.1 N HCl and analysed by Sephasorb chromatography. In rats pre-treated with NAF (0.2% in the diet over a period of 3 weeks), the concentration of N7-methylguanine in hepatic DNA (mumol/mol guanine) was 46% below control values. This is probably due to the greater amount of target DNA, as NAF caused a marked hepatomegaly with a 50% increase in total liver DNA content. Concentrations of N7-methylguanine in kidney DNA were twice as high in NAF-pre-treated animals when compared to control rats. This is unlikely to result from a shift in the metabolism of NDMA from liver to other rat tissues since the time course and extent of the conversion of [14C]NDMA to 14CO2 and 14C-labelled urinary metabolites were identical in NAF-treated and control animals. There was no indication that NAF inhibits the activity of the hepatic O6-alkylguanine-DNA alkyltransferase.

The cytotoxic action of diethyldithiocarbamate in vitro. Different inhibition of scheduled and unscheduled DNA synthesis of rat thymic and splenic cells.

Within a concentration range of 1-10 micrograms/ml, an addition of diethyldithiocarbamate (DDC) to splenic and thymic rat lymphocytes (2 X 10(6)-4 X 10(6) cells/ml) resulted in a complete inhibition of scheduled (semiconservative) DNA synthesis. Lower and higher concentrations were less effective. Under the same conditions, a strictly dose-dependent inhibition of unscheduled (excision repair) DNA synthesis, a decrease of the sedimentation rate of nucleoids, as well as changes of the thymidine pool were observed. The results suggest that the cytotoxic action of DDC in vitro may be mediated a) by the chelating properties of the drug with inhibitory effects on a variety of cellular functions, including nucleic acid precursor metabolism, b) by an immediate radiomimetic attack of the SH-group of DDC on the DNA.

[Comparative studies on the DNA synthesis of rat thymus and spleen cells in vitro under the influence of x rays, UV light and radiomimetic compounds]. / Vergleichende Untersuchungen zur DNA-Synthese von Thymus- und Milzzellen der Ratte in vitro unter dem Einfluss von Röntgenstrahlen, UV-Licht und radiomimetisch wirksamen Verbindungen.

In order to further characterize the different repairing behavior of thymus and spleen cells of rats in vitro under the influence of X-rays, UV radiation and methylmethanesulfonate (MMS), the effect of bleomycin (BM), L-cysteine (CY-E), N-ethylmaleimide (NEM), 1-beta-D-arabinofuranosylcytosine (araC), dideoxythymidine (ddT), and novobiocin (NB) on the semiconservative and restorative DNA synthesis as well as on the behavior of DNA under the alkaline elution was studied. The semiconservative DNA synthesis was inhibited by all examined agents except ddT, the restorative DNA synthesis only by NEM, araC, and NB. The stimulation of the restorative DNA synthesis was increased by UV radiation and MMS in spleen cells and by X-rays, BM and CY-E in thymus cells. Under the conditions of alkaline elution, there was a more sensitive reaction of spleen cells than of thymus cells to X-rays, BM and CY-E. The results show that thymus cells are especially qualified for the repair of short chains and spleen cells for the repair of long chains.