An Open-Label, 52-Week, Phase III Trial of Duloxetine in Japanese Patients with Chronic Low Back Pain.

OBJECTIVE: To evaluate the safety and efficacy of duloxetine treatment for 52 weeks. DESIGN: Multicenter, open-label, phase III clinical study. SETTING: Forty-one medical institutions in Japan. SUBJECTS: Japanese patients with chronic low back pain (CLBP). METHODS: Duloxetine 60 mg once-daily was administered for 52 weeks. Safety was evaluated based on adverse events (AEs), vital signs, laboratory test values, electrocardiogram, Columbia-Suicide Severity Rating Scale, and occurrence of falls. The efficacy outcome measures were the Brief Pain Inventory (BPI; average pain, worst pain, least pain, and pain right now), BPI Interference, Patient's Global Impression of Improvement (PGI-I), Clinical Global Impressions of Severity (CGI-S), Roland-Morris Disability Questionnaire-24 (RDQ-24), 36-Item Short-Form Health Survey (SF-36), and European Quality of Life-5 Dimensions Questionnaire (EQ-5D). RESULTS: In total, 151 patients (83 who completed a 14-week placebo-controlled superiority trial and 68 newly registered patients) were enrolled. The incidence rates of AEs and adverse drug reactions (ADRs) were 86.1% and 50.3%, respectively. ADRs with an incidence of ≥5% were somnolence, constipation, nausea, and dry mouth. Treatment discontinuation for AEs occurred in 16 patients. A significant reduction in the BPI average pain score (mean ± SD) was observed at all assessment time points from week 2 (-1.02 ± 1.37) to week 50 (-2.26 ± 1.63), compared with baseline. BPI pain severity (worst pain, least pain, and pain right now), BPI Interference, PGI-I, CGI-S, RDQ-24, SF-36, and EQ-5D showed significant improvement. CONCLUSION: Japanese patients with CLBP had significant pain reduction over 52 weeks without new safety concerns.

Integration of sperm DNA damage assessment into OECD test guidelines for genotoxicity testing using the MutaMouse model.

The Organisation for Economic Co-operation and Development (OECD) endorses test guidelines (TG) for identifying chemicals that are genotoxic, such as the transgenic rodent gene mutation assay (TG 488). Current OECD TG do not include assays for sperm DNA damage resulting in a critical testing gap. We evaluated the performance of the Sperm Chromatin Structure Assay (SCSA) and the Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick end Labeling (TUNEL) assay to detect sperm DNA damage within the recommended TG 488 protocol. MutaMouse males received 0, 0.5, 1, or 2 mg/kg/day triethylenemelamine (TEM), a multifunctional alkylating agent, for 28 days orally and tissues were collected two (blood) and three (sperm and bone marrow) days later. TEM significantly increased the frequency of lacZ mutants in bone marrow, and of micronuclei (MN) in both reticulocytes (%MN-RET) and normochromatic erythrocytes (%MN-NCE) in a dose-dependent manner (P < 0.05). The percentage of DNA fragmentation index (%DFI) and %TUNEL positive cells demonstrated dose-related increases in sperm (P < 0.05), and the two assay results were strongly correlated (R = 0.9298). Within the same animal, a good correlation was observed between %MN-NCE and %DFI (R = 0.7189). Finally, benchmark dose modelling (BMD) showed comparable BMD10 values among the somatic and germ cell assays. Our results suggest that sperm DNA damage assays can be easily integrated into standard OECD designs investigating genotoxicity in somatic tissues to provide key information on whether a chemical is genotoxic in germ cells and impact its risk assessment.

A systematic review on the use of cryotherapy versus other treatments for basal cell carcinoma.

BACKGROUND: Cryotherapy is a commonly discussed method for treatment of basal cell carcinoma skin cancer. Some uncertainty remains about its efficacy relative to other modalities. OBJECTIVE: To determine the efficacy and adverse events profile of cryotherapy for the treatment of basal cell carcinoma compared to other therapeutic options or non-intervention. METHODS: We systematically searched PubMed, OVID, Cochrane Library, EMBASE, CINHAL, and CANCERLIT databases for the following terms: "cryotherapy", AND "basal cell carcinoma", OR "cryosurgery" OR "cryoablation" up to April 2018. Two independent reviewers screened the results and extracted the data. Study endpoints included basal cell carcinoma recurrence, cosmetic outcome, and healing time. Study quality was assessed using the Jadad scale. RESULTS: Six clinical studies met our inclusion criteria. The efficacy and safety of cryotherapy alone or with curettage in the treatment of primary superficial and nodular basal cell carcinoma was comparable to photodynamic therapy and surgery, respectively. Cryotherapy was inferior to radiation in terms of recurrence rate. Most patients had better cosmetic outcomes with photodynamic therapy and surgery compared to cryotherapy alone, and cryotherapy with curettage. CONCLUSION: Current available data suggests equivalent efficacy of cryotherapy alone compared to photodynamic therapy or surgery, but inferior to radiotherapy. More studies are necessary to draw definitive conclusions.

A randomized trial of a low-dose Rasagiline and Pramipexole combination (P2B001) in early Parkinson's disease.

BACKGROUND: Rasagiline and pramipexole act to improve striatal dopaminergic transmission in PD via distinct and potentially synergistic mechanisms. We performed a placebo-controlled study to determine whether 2 doses of a novel slow-release, low-dose combination of rasagiline and pramipexole (P2B001) are effective and have a good safety profile in patients with early untreated PD. METHODS: Previously untreated patients with early PD were randomized (1:1:1) to once-daily treatment with P2B001 (0.3 mg pramipexole/0.75 mg rasagiline), P2B001 (0.6 mg pramipexole/0.75 mg rasagiline) or placebo in a 12-week multicenter double-blind, placebo-controlled trial. The primary endpoint was the change from baseline to final visit in Total-UPDRS score versus placebo. Secondary measures included responder analyses of patients achieving ≥4 UPDRS point reduction, and changes in Parkinson Disease Quality of Life Scale-39 and UPDRS activities of daily living and motor scores. RESULTS: A total of 149 participants were randomized and 136 (91.3%) completed the study. Adjusted mean change from baseline to final visit versus placebo in Total-UPDRS score was -4.67 ± 1.28 points for the P2B001 0.6/0.75 mg group (P = .0004) and -3.84 ± 1.25 points for the 0.3/0.75 mg group (P = .003). Significant benefits were also observed for both doses in the responder analysis (P = .0002 and P = .0001), Parkinson Disease Quality of Life Scale-39 scores (P = .05 and P = .01), and the UPDRS motor (P = .02 and P = .006) and activities of daily living (P = .005 and P = .0004) subscores. Adverse events of P2B001 were comparable to placebo apart from transient nausea and somnolence, which were more common with P2B001 treatment. CONCLUSIONS: P2B001 offers a promising treatment option for patients with early PD with good clinical efficacy and a low risk of adverse events. © 2017 International Parkinson and Movement Disorder Society.

Quantitative dose-response curves from subcellular lipid multilayer microarrays.

The dose-dependent bioactivity of small molecules on cells is a crucial factor in drug discovery and personalized medicine. Although small-molecule microarrays are a promising platform for miniaturized screening, it has been a challenge to use them to obtain quantitative dose-response curves in vitro, especially for lipophilic compounds. Here we establish a small-molecule microarray assay capable of controlling the dosage of small lipophilic molecules delivered to cells by varying the sub-cellular volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. Features with sub-cellular lateral dimensions were found necessary to obtain normal cell adhesion with HeLa cells. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. We used the surface supported lipid volume information to obtain EC-50 values for the response of HeLa cells to three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. No significant toxicity was observed on the control cells surrounding the drug/lipid patterns, indicating lack of interference or leakage from the arrays. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which we explain in terms of cell-adhesion playing a more important role in the surface-based assay. The assay should be scalable to a density of at least 10,000 dose response curves on the area of a standard microtiter plate.

Risk of subsequent malignant neoplasms in long-term hereditary retinoblastoma survivors after chemotherapy and radiotherapy.

PURPOSE: Hereditary retinoblastoma (Rb) survivors have increased risk of subsequent malignant neoplasms (SMNs). Previous studies reported elevated radiotherapy (RT) -related SMN risks, but less is known about chemotherapy-related risks. PATIENTS AND METHODS: In a long-term follow-up study of 906 5-year hereditary Rb survivors diagnosed from 1914 to 1996 and observed through 2009, treatment-related SMN risks were quantified using cumulative incidence analyses and multivariable Cox proportional hazards regression models with age as the underlying time scale. RESULTS: Nearly 90% of Rb survivors were treated with RT, and almost 40% received alkylating agent (AA) -containing chemotherapy (predominantly triethylenemelamine). Median follow-up time to first SMN diagnosis was 26.3 years. Overall SMN risk was not significantly elevated among survivors receiving AA plus RT versus RT without chemotherapy (hazard ratio [HR], 1.27; 95% CI, 0.99 to 1.63). AA-related risks were significantly increased for subsequent bone tumors (HR, 1.60; 95% CI, 1.03 to 2.49) and leiomyosarcoma (HR, 2.67; 95% CI, 1.22 to 5.85) but not for melanoma (HR, 0.74; 95% CI, 0.36 to 1.55) or epithelial tumors (HR, 0.89; 95% CI, 0.48 to 1.64). Leiomyosarcoma risk was significantly increased for survivors who received AAs at age < 1 (HR, 5.17; 95% CI, 1.76 to 15.17) but not for those receiving AAs at age ≥ 1 year (HR, 1.75; 95% CI, 0.68 to 4.51). Development of leiomyosarcoma was significantly more common after AA plus RT versus RT (5.8% v 1.6% at age 40 years; P = .01). CONCLUSION: This comprehensive quantification of SMN risk after chemotherapy and RT among hereditary Rb survivors also demonstrates an AA-related contribution to risk. Although triethylenemelamine is no longer prescribed, our findings warrant further follow-up to investigate potential SMN risks associated with current chemotherapies used for Rb.

Capture of carbon dioxide by amine-impregnated as-synthesized MCM-41.

The novel carbon dioxide (CO2) adsorbents with a high capture efficiency were prepared through impregnating the as-synthesized MCM-41 with three kinds of amines, namely diethylenetriamine (DETA), triethylenetetramine (TETA) and 2-amino-2-methyl-1-propanol (AMP). The resultant samples were characterized by small angle X-ray diffraction and low temperature N2 adsorption. The synthesis way not only saves the energy or extractor to remove the template but also is environmentally friendly due to the absence of the potential pollutants such as toluene. CO2 capture was investigated in a dynamic packed column. The sample impregnated by TETA showed the highest adsorption capacity, approximately 2.22 mmol/g at 60 degrees C due to its highest amino-groups content among the three amines. The CO2 adsorption behavior was also investigated with the deactivation model, which showed an excellent prediction for the breakthrough curves.

Visual fields in retinoblastoma survivors.

OBJECTIVE: To describe the visual field defects in retinoblastoma survivors and relate those defects to characteristics such as tumor size, tumor location, and treatment modality. METHODS: Thirty-one patients treated for retinoblastoma were included in this study. Humphrey visual fields were determined in 33 eyes. RESULTS: Twenty-seven patients (29 eyes, 68 tumors) had sufficient diagnosis and treatment data available for further analysis. Twenty-six of the 27 patients had both absolute and relative visual field defects. Four types of visual field defects were observed and correlated with location of the tumor and therapy to the individual tumors: (1) no residual defect, (2) absolute scotoma, (3) arcuate and sector scotoma, and (4) "pseudo"-visual field defects caused by relative enophthalmos resulting from radiation. CONCLUSIONS: Patients with retinoblastoma demonstrate a variety of long-term visual field defects after treatment for their intraocular disease. Characteristics that determine the size and type of defects are tumor size, tumor location, and treatment method.

Nonocular second primary tumors after retinoblastoma: retrospective study of 111 patients treated by electron beam radiotherapy with or without TEM.

The objective of this study was to assess the role of various clinical and treatment factors involved in the long-term incidence of nonocular second primary tumors following retinoblastoma. The study was based on 111 patients treated between 1963 and 1977 according to the same radiotherapy protocol (electron beam radiotherapy) alone or in combination with triethylene melamine (TEM). Various statistical methods were used to obtain the actuarial survival curve, the cumulative incidence of second primary tumors, and comparisons of patient groups and subgroups. The 5-, 10-, 20-, and 30-year survival rates were 75%, 70%, 63%, and 55% with a follow-up of 23 to 35 years. The study reports the various parameters concerning 111 children and 17 second primary tumors: sex, age at treatment, histology of the retinoblastoma and second primary tumors, site of second tumors (anatomic and compared with irradiation field), radiation dose, time to onset, and chemotherapy with or without TEM. The results are discussed and compared with the data reported in the literature. Electron beam radiotherapy at a dose of 45 Gy does not eliminate the risk of nonocular second primary tumors. TEM also does not modify survival or the overall incidence of second primary tumors, but significantly increases the risk of second primary tumors outside the irradiation field.

Adaptive resistance of Saccharomyces cerevisiae to chronic treatment with genotoxic and nongenotoxic carcinogens.

The exposure of mammalian cells or tumors for weeks or months to low nonlethal doses of cytostatic drugs may induce multidrug resistance, which can be enhanced by a variety of DNA-damaging agents. Multidrug resistance to a variety of drugs has been observed. But in yeast, DNA-damaging agents have not yet been tested. As the appearance of resistance is the result of longterm exposure, we decided to extend the application of test substances to a period of up to 400 days. In such long-term experiments S. cerevisiae MP1 adapted to treatment with low doses of mutagens. Consistent results were obtained for both genotoxic and nongenotoxic carcinogenic substances, which implies that there may be a single pathway for carcinogens with different modes of action.

Anti-recombinogenic and convertible co-mutagenic effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted pyrimidine nucleoside analogs in S. cerevisiae MP1.

In experiments using yeast, without addition of an external metabolic activation system, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was co-mutagenic and showed an insignificant anti-recombinogenic effect in combination with triethylene melamine (TEM). In the presence of activating S9-mix, the anti-recombinogenicity and co-mutagenicity could clearly be seen. At higher concentrations the co-mutagenic effect was converted into anti-mutagenicity. The other three 5-substituted pyrimidine nucleoside analogs were tested only in the presence of activating S9-mix and showed similar effects. As TEM is a direct alkylating agent that is inactivated by liver microsomes, the higher activity in presence of S9-mix can be interpreted as resulting from metabolic activation of the 5-substituted pyrimidine nucleoside analogs. In previous experiments using yeast bacteria, Drosophila or mice, tumor promoters were co-recombinogenic/anti-mutagenic, and co-carcinogens were co-mutagenic/anti-recombinogenic. Thus, there is not only an operational difference between tumor promoters and co-carcinogens but a real difference in respect to their genetic effectiveness. As up to now only co-carcinogens have shown co-mutagenic and anti-recombinogenic effects, it is perhaps possible that, within a certain concentration range, 5-substituted pyrimidine nucleoside analogs may have co-carcinogenic activity in carcinogenicity tests. At higher concentrations the co-carcinogenic effect may be converted into an anti-carcinogenic one.

Comparison of the mutagenicity and mutagen specificity of ethylenimine with triethylenemelamine in the ad-3 region of heterokaryon 12 of Neurospora crassa.

Studies have been performed to compare the mutagenicity and mutagenic specificity of the trifunctional alkylating agent, triethylenemelamine (TEM), and a closely related monofunctional agent, ethylenimine (EI), in the adenine-3 (ad-3) region of a 2-component heterokaryon (H-12) of Neurospora crassa. The primary objective of our studies was to characterize the genetic damage produced by each agent with regard to (1) mutagenic potency, and (2) the spectrum of specific-locus mutations induced in a lower eukaryotic organism. As in higher eukaryotes, specific-locus mutations in the ad-3 region of H-12 result from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations. Specific-locus mutations resulting from gene/point mutation and multilocus deletion mutation can be detected in higher eukaryotes, but multiple-locus mutations can be detected only with difficulty or not at all. Our experiments with the ad-3 forward-mutation assay have demonstrated that TEM is a strong mutagen (maximum forward-mutation frequency between 100 and 1000 ad-3 mutations per 10(6) survivors) and EI is a moderate mutagen (maximum forward-mutation frequency between 10 and 100 ad-3 mutations per 10(6) survivors) for the induction of specific-locus mutations in the ad-3 region. Classical genetic tests were used to identify the different genotypic classes and subclasses among the EI- and TEM-induced ad-3 mutations from each experiment. The overall data base demonstrates that both EI- and TEM-induced ad-3 mutations result predominantly from gene/point mutations at the ad-3A and ad-3B loci (97.3% and 95.5%, respectively), and infrequently from multilocus deletion mutations (2.7% and 4.5%, respectively). Heterokaryon tests for allelic complementation on TEM- and EI-induced ad-3B mutations, however, have revealed a difference between the percentages showing allelic complementation (63.1% and 40.9%, respectively). Based on the specific revertibility of complementing and noncomplementing ad-3B mutations induced by other agents, this difference in the percentages of ad-3B mutations showing allelic complementation results from a difference between the spectrum of genetic alterations at the molecular level. In addition, comparison of the ratio of TEM-induced ad-3A and ad-3B mutations with those induced by EI has revealed a difference between the ad-3B/ad-3A ratios. Additional comparisons are made of the mutagenic effects of TEM and EI with those of other chemical mutagens and carcinogens in the ad-3 specific-locus assay in Neurospora.

Triethylenemelamine: induction of specific-locus mutations in the ad-3 region of heterokaryon 12 of Neurospora crassa.

The mutagenicity of the trifunctional alkylating (or cross-linking) agent TEM (triethylenemelamine or 2,4,6-tris(1-aziridinyl)-1,3,5-triazine) in the adenine-3 (ad-3) region was studied with a two-component heterokaryon (H-12) of Neurospora crassa. The objective was to characterize the genetic damage produced by this chemical to determine the spectrum of specific-locus mutations induced in a lower eukaryotic organism and to compare this spectrum with that induced in the mouse. Specific-locus mutations in the ad-3 region of strain H-12 result from gene/point mutations, multiple-locus mutations, and multilocus deletion mutations at the closely linked ad-3A and ad-3B loci. These loci control two sequential biochemical reactions in the purine biosynthetic pathway. A 0.1 M solution of TEM was used to treat conidial suspensions of H-12 for 20, 40, 80, 120, or 170 min to obtain dose-response curves for (1) inactivation of conidia, and (2) the induction of specific-locus mutations in the ad-3 region. These experiments demonstrated that TEM is a strong mutagen (maximum forward-mutation frequency between 100 and 1000 ad-3 mutations per 10(6) survivors) for the induction of specific-locus mutations in the ad-3 region. Both biochemical and classical genetic tests were used to characterize the TEM-induced ad-3 mutations from each of the five treatment groups to distinguish between the different genotypic classes and subclasses. The overall data base from these genetic studies demonstrates that TEM-induced ad-3 mutations result predominantly (95.5% [769/805]) from gene/point mutations at the ad-3A and ad-3B loci, and from a low percentage (4.5% [36/805) of multilocus deletion mutations. In addition, TEM induces an unusually high frequency of multiple-locus mutations with sites of recessive lethal damage closely linked with the ad-3 region. Comparison of the dose-response curves for the major classes and subclasses of TEM-induced ad-3 mutations demonstrates (1) that gene/point mutations and multilocus deletion mutations increase as the 1.4 power of TEM treatment time, and (2) that the two classes of TEM-induced multiple-locus ad-3 mutations consisting of gene/point mutations with separate sites of recessive lethal damage increase at about the 1.96 power of TEM treatment time. When the data from the present specific-locus studies are compared with those in the mouse, we find, insofar as such comparisons are possible, that a similar spectrum of specific-locus mutations has been induced by TEM in each assay system.

Chromosome analysis of human spermatozoa exposed to antineoplastic agents in vitro.

We studied in vitro the cytogenetic effects of six antineoplastic agents, bleomycin (BM), cyclophosphamide (CP), daunomycin (DM), methyl methanesulfonate (MMS), mitomycin C (MMC) and triethylenemelamine (TEM) on spermatozoa, using an interspecific in vitro fertilization system between zona-free hamster oocytes and human or bull spermatozoa. In preliminary experiments with bull spermatozoa, clastogenic effects were clearly shown with BM, DM, MMS and TEM, but not with CP and MMC. In main experiments, the effects of the first four chemicals were studied in detail with human spermatozoa. Total numbers of 585 and 512 spermatozoa were karyotyped in the control and the chemical-treated groups respectively. The incidence of spermatozoa with structural chromosome aberrations was 34.5%, 53.0%, 59.3%, and 55.6% in the BM (50 micrograms/ml, 90 min), DM (0.1 microgram/ml, 90 min), MMS (100 micrograms/ml, 120 min) and TEM (0.1 micrograms/ml, 120 min) groups respectively, each showing a significantly higher incidence than the matched controls (10.1-13.5%). Breakage-type aberrations were more frequent than exchange-type aberrations in the BM, MMS and TEM groups, while the exchange-type aberrations were more frequent in the DM group. Exchanges were mainly of the chromatid type in the DM, MMS and TEM groups, while chromosome-type exchanges occurred more frequently in the BM group. These results are discussed in relation to previous data on chemical-induced chromosome aberrations in mammalian somatic cells and in mouse spermatozoa.

Measurement of DNA integrity and structure in Xenopus embryos in the presence of hydroxyurea, actinomycin-D, and triethylenemelamine using the fluorescent probe Hoechst 33258.

Cell health assay of water quality (CHAWQ) is an assay using intracellular biomarkers measured by optical techniques. CHAWQ uses embryos of the South African clawed frog, Xenopus laevis, and optical transducers of intracellular biomarkers to obtain rapid assessment of toxicity to frog embryos. Since the biomarkers are common to all cells, CHAWQ can indicate toxicity of different classes of chemicals. Among the biomarkers used are 1) the change in synthesis rate, 2) the structure, or 3) the environment of DNA. Measurement of DNA to detect genotoxicants has previously used extracted DNA or flow cytometry to detect alterations in DNA content or configuration. We report the use of viable frog embryos and the fluorescent probe Hoechst 33258 to detect the effect of three DNA-active chemicals--actinomycin-D, hydroxyurea, and triethylenemelamine (TEM)--on DNA in intact embryos. We found that we can detect changes in the DNA in the presence of toxicants at concentrations comparable to longer-term assays but following a much shorter time of drug exposure. Actinomycin-D caused a fluorescence decrease, TEM caused a fluorescence increase, whereas hydroxyurea gave a biphasic response. Hydroxyurea caused a decrease at low concentrations and an increase at higher concentrations. Concentration-response data for TEM, hydroxyurea, and actinomycin-D generated EC50 values of 0.1 mg/ml, 1.4 mg/ml, and 6.34 micrograms/ml, respectively.

Further flow cytometric studies of the effects of triethylenemelamine on somatic and testicular tissues of the rat.

Exposure to the mutagen triethylenemelamine on rat bone marrow, blood, and testis was studied using flow cytometry of DAPI-stained nuclei. Increased coefficients of variation (CVs) of the G1 peaks were observed in bone marrow and blood after both 1 d and 5 d exposures. After 5 d exposure and 7 d recovery both tissues had recovered, in some cases to significantly lower CVs. Increased CVs of the 1C peak of testis were observed only after 5 d exposure to the high dose with no subsequently observed recovery. Bone marrow cells also were stained with Hoechst 33258 and Propidium Iodide. No differences among dyes were observed indicating that increased CVs likely are due to DNA damage resulting from interactions with the mutagen rather than differences in how the dyes bind to DNA relative to mutagen binding. This study demonstrates that differences occur among tissues in how quickly they respond and recover from mutagen exposure. Increased CVs, cell cycle alterations, and decreased CVs after recovery are all potentially useful biomarkers of effect for laboratory and field studies in environmental toxicology.

Sister-chromatid exchanges in Picea abies--a test for genotoxicity in forest trees.

A genotoxicity test, based on the evaluation of sister-chromatid exchange frequencies, has been developed for the spruce fir. The basic frequency was 36.9 SCEs/cell. Mitomycin C treatment (MMC, 5 x 10(-6) M, 0.5 h) doubled the 'spontaneous' SCE frequency, maleic hydrazide treatment (MH, 5 x 10(-4) M, 0.5 h) increased it nearly 7-fold. This corresponds to data obtained previously for Vicia faba. Chromatid-type aberrations were induced by the same mutagens (MH, 5 x 10(-4) M, 0.5 h; MMC 2 x 10(-5) M, 1 h) or by triethylenemelamine (TEN, 2 x 10(-4) M, 0.5 h). MH treatment resulted in aberration yields comparable to those observed in Vicia faba, MMC and TEM were less efficient aberration inducers in P. abies. While SCEs may be counted for single chromosomes, for reliable evaluation of chromatid aberrations large numbers of complete and well spread metaphases have to be inspected.

Combined application of MH and TEM for conditioning treatment simultaneously triggers two protective functions against induction of chromatid aberrations in Vicia faba root tip meristem cells.

Conditioning pre-exposure of Vicia faba root tip meristem cells to triethylenemelamine (TEM) does not trigger an adaptive response to maleic hydrazide (MH) and vice versa. Since TEM conditioning treatment can induce protective effects (as evident from the yield of metaphases with chromatid aberrations) against TEM challenging treatment and MH conditioning can trigger an adaptive response to MH challenging treatment, two different protective functions are apparently triggered in dependence on the agent used for conditioning pre-exposure. When a mixture of TEM and MH is being used for conditioning treatment, adaptive responses to both TEM and MH can simultaneously be induced and significantly reduce the yield of metaphases with chromatid aberrations observed after challenge treatment with TEM or MH.

Inhibition of protein synthesis by cycloheximide does not prevent adaptive responses triggered by heavy metal salts in Vicia faba.

Copper sulfate, lead nitrate, and cadmium nitrate conditioning treatments of Vicia faba root tip meristem cells result in adaptive responses to triethylenemelamine (TEM)- and maleic hydrazide (MH)-induced genotoxic effects, i.e., they significantly reduce the yield of metaphases with chromatid aberrations. Contrary to adaptive responses triggered by low clastogen concentrations or heat shock, protective effects induced by heavy metals cannot be prevented by inhibition of protein synthesis and last for a much longer time span (up to at least 48 h). These and other data support the conclusion that the cells can react to the impact of external factors by various inducible functions which eventually protect their DNA from damage.

Low temperature between conditioning and challenge treatment prevents the 'adaptive response' of Vicia faba root tip meristem cells.

When the temperature during intertreatment time (2 h) between conditioning and challenge treatment of Vicia faba root tip meristems with either triethylenemelamine or maleic hydrazide was reduced from 24 degrees C to 12 degrees C no adaptive response occurred any more. The yield of metaphases with chromatid aberrations under these circumstances was similar to that observed after challenge treatment alone, i.e., no reduction occurred. This indicates that the metabolic state of the cells is of critical importance for the presence or absence of adaptive responses.