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-7H-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.

Bioactivation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to reactive intermediates that bind covalently to macromolecules and produce genotoxicity.

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

Spontaneous and ethylnitrosourea-induced mutation fixation and molecular spectra at the lacI transgene in the Big Blue rat-2 embryo cell line.

Big Blue Rat-2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]-induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 micrograms ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 X 10(5). Dose-related increases in mutant frequency were observed following ENU treatment. Kinetics (time course) of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 micrograms ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C-->A:T transitions (five at CpG sites), six (23%) G:C-->T:A transversions, three (11.5%) G:C-->C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion+insertion) mutation. Among 46 ENU-induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C-->A:T transitions (four at CpG sites), five (13.5%) A:T-->G:C transitions, four (10.8%) G:C-->T:A transversions, 11 (30%) A:T-->T:A transversions, and two (5.4%) A:T-->C:G transversions. Nearly 50% of the base substitutions in the ENU-treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU-induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 micrograms/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity.

Risk and benefit evaluation in development of pharmaceutical products.

Pharmaceutical products are intended to cure disease, reduce pain and suffering, prolong life, and correct metabolic deficits in patients. However, the potential patient population is intrinsically genetically heterogenous, and this factor complicates the evaluation of data on all aspects of safety evaluation of new drugs. Often the genetic heterogeneity is related to drug metabolizing capacity, but recent evidence suggests that heterogeneity in repair capacity as well as structural integrity of the chromatin (fragile X) have been shown to be relevant. Because drugs are biologically active and may have more than one type of effect, the evaluation of a large number of parameters is necessary in arriving at a rational estimate of potential risk. In this paper, several specific examples of risk assessments and some generic genotoxicity questions that are recurrent, including the question of the relevance of in vitro chromosomal aberration induction at high dose/sampling time, are raised. Other examples of the kinds of concerns from the safety evaluation of U-48753E, U-54461, and U-68,553B are discussed. The drug U-48753E was discovered to be slightly mutagenic in the AS52 assay, and significant efforts were expended in evaluation of the metabolism-based generation of a reactive intermediate. The drug U-54,461 was shown to be capable of breaking chromosomes in vitro but extensive in vivo data as well as a variety of other studies served to reduce the level of concern substantially.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased PKA and PKC activities accompany neuronal differentiation of NT2/D1 cells.

After retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (C alpha and C beta) subunits of PKA increased after differentiation. Total PKA activity levels increased 7-fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12-fold induction of Type 2 (beta) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (beta) and Type 3 (alpha) PKC protein levels also accompanied differentiation. These changes in PKA- and PKC-specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state in these cells.

Transfection of a mutant regulatory subunit gene of cAMP-dependent protein kinase causes increased drug sensitivity and decreased expression of P-glycoprotein.

Wild-type Chinese hamster ovary (CHO) cells were transfected with a DNA clone (MT-REV, site A) carrying a mouse gene for a dominant mutant regulatory subunit (RI) gene of cAMP-dependent protein kinase (PKA) from S49 cells along with a marker for G418 resistance. G418-resistant transfectant clone R-2D1 was resistant to 8-Br-cAMP-induced growth inhibition and morphological changes. The cells also did not phosphorylate a 50-kDa protein after cAMP stimulation and had decreased PKA activity, both characteristics of PKA mutants. Northern blot analysis indicated that clone R-2D1 was actively transcribing the MT-REV (site A)-specific RNA. We also tested clone R-2D1 for sensitivity to certain natural product hydrophobic drugs and found increased sensitivity to several drugs including adriamycin. Hypersensitivity to these drugs has previously been shown by us to be a characteristic of a CHO PKA mutant cell line. Expression of the mutant RI gene is also associated with a decrease in expression of the multidrug resistance associated P-glycoprotein (gp170) mRNA and protein. These results show that the PKA mutant RI gene from S49 cells acts as a dominant mutation to reduce the total PKA activity in the CHO transfectants as it does in mouse S49 cells. This study also confirms that reduced PKA activity modulates the basal multidrug resistance of these cells, apparently by causing decreased expression of the mdr gene at the protein and mRNA level.

Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard.

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.

Increased amount of a 25-kilodalton phosphoprotein after v-mos transfection of CHO cells.

We transfected Chinese hamster ovary (CHO) cells with a cloned v-mos gene (pHT25). The mos family of oncogenes has previously been shown to have serine-threonine kinase activity. This kinase activity may be required for oncogenic transformation, although its exact biological role is unknown. We found that the transfected cells had an altered morphology, a slower doubling time, and an apparent increase in the amount of a 25-kilodalton (kDa) phosphoprotein that appeared to be of low abundance. Transfection of CHO cells with a cloned temperature-sensitive mos gene (ts159) led to isolation of a cell line that showed the presence of the 25-kDa phosphoprotein at the permissive but not at the nonpermissive temperature, suggesting a direct relationship between mos activity and the presence of this phosphoprotein. The characteristics of altered morphology and depressed growth rate were reminiscent of changes seen after the activation of the cyclic AMP-dependent protein kinase (PKA) in CHO cells. However, PKA activation did not stimulate phosphorylation of this 25-kDa protein, nor was there a change in total PKA activity in these cells. We suggest that the increased presence of the 25-kDa phosphoprotein is a consequence of the v-mos transfection and that it may be involved in the change of morphology and growth rate seen in the CHO cells. Phosphorylation of this protein may be a useful marker of mos and have some functional importance in the transformation of cells by the v-mos oncogene.

Distance education: some issues.

PIP: Distance teaching has increased access to education and democratized educational opportunities in many countries, but the question arises as to at what cost and with what degrees of effectiveness. The proliferation and rapid growth of distance teaching systems in the developing world have outstripped research that compares the cost effectiveness of distance teaching with that of traditional programs. Early indications from the major Asian open universities, in China, Indonesia, and Thailand, suggest that costs per student are substantially lower than conventional alternatives, but dropout rates have been on the average 30-50%. In some areas, student services have been nonexistent. Further, as enrollments increase, administrative control and logistical support tend to deteriorate. Due to this, distance education systems risk being identified as 2nd class alternatives to established academic institutions. Key staff may be overworked and underpaid, and demands for course development generally exclude time for research or for direct contact with students. The value and marketability of the certificates awarded by distance learning institutions continue to be challenged by traditional educators and other critics who perceive distance education institutions as diploma mills. In regard to the roles assigned to communication media, these vary widely within distance learning systems. There are distinct advantages to incorporating broadcast media within any distance teaching strategy. Radio and television can carry the major instructional burden, yet most often they are employed in a supplemental role with distance education programs. The effectiveness of the media depends on the amount of time, content, expertise, and formative evaluation that are brought to bear on course design. The hallmark of broadcast education continues to be its distributive power and its ability to reach large numbers of learners.^ieng

Mutagenicity of rat-liver S9 to L5178Y mouse lymphoma cells.

Rat-liver S9 preparations became highly mutagenic to cultured L5178Y mouse lymphoma cells when the exposure period was increased to 18-24 h or when S9 mix was preincubated in Fischer's medium at 37 degrees C for 19 h and then used to treat the cells for 4 h. Five different S9 preparations (from untreated and Aroclor 1254-treated Fischer 344 or Sprague-Dawley male rats) behaved similarly. S9 mix, which contained 1 mM NADP and 5 mM isocitrate as cofactors, was more mutagenic than S9 alone. Heat treatment of S9 did not destroy its mutagenic activity, but the addition of cofactors no longer stimulated an increase in mutagenicity, as observed with native S9. Treatment with cofactors was not mutagenic. These results implied the involvement of both energy-independent and NADPH-dependent enzymatic changes in S9 mix in producing mutagenic substances. The mutagenic treatments with S9 or S9 mix induced predominantly small TFT-resistant mutant colonies, which suggested that these treatments should be clastogenic to cultured mammalian cells. A warning was given that test chemicals evaluated as mutagenic only in the presence of S9 mix may instead be accelerating the decomposition of S9 mix into mutagens, and it may become necessary to experimentally distinguish between these two mechanisms before a chemical can be regarded as mutagenic.

Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs.

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.

Phorbol myristate acetate inhibits growth in S49 cells: isolation of resistant variants.

We have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild-type (wt) cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA-induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild-type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild-type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.

Radio Santa Maria: a case study of participatory evaluation.

PIP: Radio Santa Maria (RSM), established by the Catholic Church in the Dominican Republic in 1964, broadcasts programs leading to certificates at the primary and intermediate levels and stresses education as a tool to help individuals cope with their environment. This article describes the history of an evaluation of RSM based on the participation of RSM personnel. RSM staff carried out the field work, while members of the expatriate evaluation team helped to design the survey instruments, trained staff in evaluation techniques, and conducted the statistical analyses. A 1975 policy document served as the basis of the evaluation. An important advantage of the self-study approach was that the evaluation process itself resulted in program improvements during the study period. As active participants in the process, RSM staff developed important evaluation skills and a computerized information retrieval system was installed. A disadvantage of the participatory approach to program evaluation is the time required to build personnel relations and transfer technical skills. Compromises may be necessary that undermine professional standards and reduce data reliability. On the other hand, self-study can provide data of more depth than external evaluations. It can overcome the neglect of inputs and processes that characterizes traditional output-oriented evaluations. It is concluded that if the purpose of an evaluation is to improve an organization, it is useful to involve the entire organization in the process.^ieng