Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing.

Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.

Mutation spectrum of o-aminoazotoluene in the cII gene of lambda/lacZ transgenic mice (MutaMouse).

The o-aminoazotoluene (AAT) has been evaluated as a possible human carcinogen by the International Agency for Research on Cancer. In rodents, it is carcinogenic mainly in the liver, and also in lung following long term administration. We previously examined in lambda/lacZ transgenic mice for the induction of lacZ mutations in liver, lung, urinary bladder, colon, kidney, bone marrow, and testis. AAT induced gene mutations strongly in the liver and colon. In the present report, we reveal the molecular nature of mutations induced by AAT in the lambda cII gene (the cII gene, a phenotypically selectable marker in the lambda transgene, has 294bp, which makes it easier to sequence than the original target, the 3kb lacZ gene). The cII mutant frequency in liver and colon was five and nine times higher, respectively, in AAT-treated mice than in control mice. Sequence analysis revealed that AAT induced G:C to T:A transversions, whereas spontaneous mutations consisted primarily of G:C to A:T transitions at CpG sites.

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse).

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.

Substituent effect of a fluorine atom on the mutagenicity of nitroquinolines.

Some 16 nitroquinolines (NQs) and their fluorinated derivatives were tested for mutagenicity in Salmonella typhimurium TA100 without S9 mix to investigate the effect of fluorine-substitution on the mutagenicity. These NQs consist of 5-NQs, 5-nitroquinoline N-oxides (5-NQOs), N-methyl-5-nitroquinolinium methanesulfonates (N-Me-5-NQs) and 8-NQs, including three ortho-F-NQs, one meta-F-NQ, four para-F-NQs and four 3-F-NQs. For this purpose, eight F-NQs were newly synthesized. The data indicated that the ratio of the mutagenic activities (revertants/plate/nmol) of fluorinated NQs to those of the corresponding parent non-fluorinated compounds ranged from 0.6- to 119-fold. The fluorine atom located para to the nitro group markedly enhanced the mutagenicity (24-fold and more), while three ortho-fluorinated derivatives showed no significant increase in mutagenicity (enhancement ratio were 0.6, 0.8 and 1.7). With respect to 8-NQs, its meta-fluorinated derivative also had an enhanced mutagenicity over the parent compound (53-fold). In addition, although N-Me-5-NQ was less mutagenic than 5-NQ and 5-NQO, the mutagenicity of N-Me-5-NQ was most significantly enhanced by fluorine-substitution. These results suggest that introduction of a fluorine atom to the molecule in question may be a useful tool to modify their mutagenic potency and to better understand the mechanism of mutation.

Cytotoxic factor induced in murine serum after intravenous administration of a dehydrogenation polymer of p-coumaric acid (a synthetic lignin).

A cytotoxic factor (CF) toward cultured murine leukemia L1210 cells was induced in mouse serum by intravenous injection of a dehydrogenation polymer of p-coumaric acid (DHP-pCA). When the serum from the treated mice was diluted with ethanol, CF was preserved in its supernatant (EtOH-sup). An EtOH-sup prepared from untreated control mice also showed cytotoxicity, although at much higher concentrations. The CF activity of EtOH-sups from both treated and untreated mice was completely eliminated by acid treatment at pH 2 at 90 degrees C for 30 min but kept intact by alkali treatment. In addition, the CF activity of both EtOH-sups was not affected by digestion with chymotrypsin. CF was recovered in a neutral MeOH-eluate from a DEAE-cellulofine column but not in HCI-MeOH eluate, in which lignified materials including DHP-pCA should have been recovered. These findings strongly suggest that CF is not a metabolite of DHP-pCA but an endogenous component of the normal serum which is augmented by DHP-pCA administration.

In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist.

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.

Diversity of calcium signaling by metabotropic glutamate receptors.

During prolonged application of glutamate (20 min), patterns of increase in intracellular Ca2+ concentration ([Ca2+]i) were studied in HEK-293 cells expressing metabotropic glutamate receptor, mGluR1alpha or mGluR5a. Stimulation of mGluR1alpha induced an increase in [Ca2+]i that consisted of an initial transient peak with a subsequent steady plateau or an oscillatory increase in [Ca2+]i. The transient phase was largely attributed to Ca2+ mobilization from the intracellular Ca2+ stores, but the sustained phase was solely due to Ca2+ influx through the mGluR1alpha receptor-operated Ca2+ channel. Prolonged stimulation of mGluR5a continuously induced [Ca2+]i oscillations through mobilization of Ca2+ from the intracellular Ca2+ stores. Studies on mutant receptors of mGluR1alpha and mGluR5a revealed that the coupling mechanism in the sustained phase of Ca2+ response is determined by oscillatory/non-oscillatory patterns of the initial Ca2+ response but not by the receptor identity. In mGluR1alpha-expressing cells, activation of protein kinase C selectively desensitized the pathway for intracellular Ca2+ mobilization, but the mGluR1alpha-operated Ca2+ channel remained active. In mGluR5a-expressing cells, phosphorylation of mGluR5a by protein kinase C, which accounts for the mechanism of mGluR5a-controlled [Ca2+]i oscillations, might prevent desensitization and result in constant oscillatory mobilization of Ca2+ from intracellular Ca2+ stores. Our results provide a novel concept in which oscillatory/non-oscillatory mobilizations of Ca2+ induce different coupling mechanisms during prolonged stimulation of mGluRs.

Kainate excitotoxicity is mediated by AMPA- but not kainate-preferring receptors in embryonic rat hippocampal cultures.

We investigated kainate-induced excitotoxicity in embryonic rat hippocampal cells cultured in a chemically defined medium. Treatment with kainate for 24 h resulted in neuronal death, as assessed by the release of lactate dehydrogenase into the culture media. This neurotoxic effect was kainate dose- and culture age-dependent. EC50 of kainate was 127 +/- 11 microM. 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (f)quinoxaline (NBQX) completely blocked the toxicity, while MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, also blocked it but not completely. Furthermore, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) attenuated the kainate injury, while the selective and noncompetitive AMPA-preferring receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzo-diazepine (GYKI 52466) blocked it completely. Concanavalin A (ConA), which potentiates the response to kainate at kainate-preferring receptors, had little effect on kainate toxicity. Further, AMPA alone induced little toxicity, but produced remarkable toxicity when cyclothazide was used to block the desensitization of AMPA-preferring receptors. These results indicate that kainate excitotoxicity in hippocampal cultures is mediated by AMPA- but not kainate-preferring receptors, and that it involves NMDA-receptor-mediated toxicity. The non-desensitizing response at AMPA-preferring receptors may play an important role in kainate-induced excitotoxicity.

The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development.

Previous studies have shown that recessive mutations at the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON1 (LEC1) loci lead to various abnormalities during mid-embryogenesis and late embryogenesis. In this study, we investigated whether these loci act in independent regulatory pathways or interact in controlling certain facets of seed development. Several developmental responses were quantified in abi3, fus3, and lec1 single mutants as well as in double mutants combining either the weak abi3-1 or the severe abi3-4 mutations with either fus3 or lec1 mutations. Our data indicate that ABI3 interacts genetically with both FUS3 and LEC1 in controlling each of the elementary processes analyzed, namely, accumulation of chlorophyll and anthocyanins, sensitivity to abscisic acid, and expression of individual members of the 12S storage protein gene family. In addition, both FUS3 and LEC1 regulate positively the abundance of the ABI3 protein in the seed. These results suggest that in contrast to previous models, the ABI3, FUS3, and LEC1 genes act synergistically to control multiple elementary processes during seed development.

Characterization of a cytotoxic factor induced in murine serum after the intravenous administration of dehydrogenation polymers of phenylpropenoids (a class of synthetic lignins).

A cytotoxic factor (CF) appeared in murine serum after the intravenous injection of the dehydrogenation polymers (DHPs) of p-coumaric acid (DHP-pCA), caffeic acid (DHP-CA), and ferulic acid (DHP-FA), which are categorized as a class of synthetic lignins. The highest CF activity was observed 15 min after the i.v. injection of DHP-pCA. CF is likely to be cytocidal through an apoptotic mechanism accompanied by nucleosome-sized DNA fragmentation. CF is extractable with aqueous ethanol and highly stable against heat, proteases, and acid/alkali treatments. The ethanol extract showed cytotoxicity toward various cultured cell lines and also ascites carcinoma cells in vivo. The parent molecules DHPs did not show any appreciable cytotoxicity. After the induction of CF activity, the activity quickly diminished and completely disappeared from the blood stream within an hour or so. The cytotoxicity was observed only when the target cells were exposed to CF for longer than 10 h.

Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization.

Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study. The Y-H58-F and R-H100b-A mutants possessed catalytic activities comparable to that of the wild-type, and the Y-H58-H and Y-L32-F mutant displayed an approximately fivefold decrease in k(cat)/Km. In the transition state analysis, the plots of logK(TSA) versus log(k(cat)/Km) for the mutants are linear, with a slope of approximately 1.0, indicating that the entire hapten-binding energy in the mutants is also utilized to bind the transition state and to accelerate the catalysis. In addition, a dramatic change in the catalytic activity was observed when the histidine residue (27d) in the CDR1 light chain was replaced with alanine. The H-L27d-A mutant had no detectable catalytic activity. This mutation led to a large, 40-fold reduction in transition state binding, with no change in substrate binding. Coupled with the previous kinetic studies and chemical modifications of the intact 6D9 antibody, this mutagenesis study has demonstrated that His L27d plays an essential role in stabilization of the transition state, the mechanism of catalysis by the 6D9 antibody.

Enhanced cytotoxicity of alkyl viologens and N,N'-diamino analogs toward cultured murine leukemia L1210 cells under vortex-stirring with a high molecular weight polyacrylic acid.

Alkyl viologens showed cytotoxicity when incubated with cultured murine leukemia L1210 cells for 48 h, whereas they were not cytotoxic when briefly incubated for 10 min. Under the permeabilizing conditions achieved by vortex-stirring with a high molecular weight polyacrylic acid (A-119), appreciable cytotoxicity was shown even after a 10-min exposure to any of the alkyl and amino viologens examined. Acetylamino viologen showed no cytotoxicity regardless of the presence or absence of A-119. This permeabilizing procedure was demonstrated to be applicable to internalize water-soluble positively charged viologen molecules into the cell.

Control of calcium oscillations by phosphorylation of metabotropic glutamate receptors.

Stimulation of two metabotropic glutamate-receptor subtypes, mGluR1 and mGluR5, triggers the release of Ca2+ from intracellular stores through the inositol-(1,4,5) trisphosphate (InsP3) pathway. Here we report that glutamate induces single-peaked intracellular Ca2+ mobilization in mGluR1alpha-transfected cells but elicits Ca2+ oscillations in mGluR5a-transfected cells. The response patterns of the intracellular Ca2+ increase depend upon the identity of a single amino acid, aspartate (at position 854) or threonine (at position 840), located within the G-protein-interacting domains of mGluR1alpha and mGluR5a, respectively. Pharmacological and peptide mapping analyses indicated that phosphorylation of the threonine residue at position 840 of mGluR5a by protein kinase C (PKC) is responsible for the generation of Ca2+ oscillations in mGluR5a-expressing cells. To our knowledge this is the first evidence that PKC phosphorylation of G-protein-coupled receptors is important in producing oscillations in intracellular Ca2+ signalling.

Characterization of YM90K, a selective and potent antagonist of AMPA receptors, in rat cortical mRNA-injected Xenopus oocytes.

The inhibitory potencies of 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K), 2-3,dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiazep ine (GYKI 52466) at excitatory amino acid receptors were examined in rat cortical mRNA-injected Xenopus oocytes using a two-electrode voltage clamp. Schild analysis of YM90K and NBQX inhibition of kainate currents yielded pA2 values of 6.83 +/- 0.01 and 7.24 +/- 0.01, respectively. GYKI 52466 reduced the maximum kainate response and increased the kainate EC50 in a dose-dependent manner, suggesting that the antagonism of AMPA receptors by GYKI 52466 is mixed competitive and non-competitive for kainate. Schild analysis of YM90K and NBQX inhibition of kainate currents in the presence of 30 microM cyclothiazide yielded pA2 values of 6.62 +/- 0.03 (slope: 1.02 +/- 0.01) and 7.10 +/- 0.02 (slope: 1.00 +/- 0.02), respectively, consistent with competitive antagonism. Cyclothiazide potentiated the AMPA response as well as the kainate response and increased the apparent Hill coefficients in a concentration-dependent manner. The potency of YM90K to inhibit AMPA-induced current could be reduced by increasing the concentration of cyclothiazide. We showed that YM90K is a potent and competitive antagonist for AMPA receptors and the apparent affinity of competitive antagonists was reduced by cyclothiazide. Cyclothiazide can affect the interaction between receptors and both agonists and antagonists, suggesting that it might allosterically alter the affinity of agonists and competitive antagonists for their binding site on the AMPA receptor complex.

Processing and secretion of human growth hormone with an artificial signal sequence.

We examined the secretion of human growth hormone in yeast cells with the artificial signal sequence L8LP, which is functional for human lysozyme secretion. The precursor was cleaved efficiently and the mature protein was secreted into the periplasmic space, but the protein aggregated. These results suggest that L8LP is also functional for human growth hormone. pI precipitation might be responsible for the aggregation.

Conformation and length of the signal sequence affect processing of secretory protein.

Processing of human lysozyme with artificially designed signal sequences was examined in an in vitro translation-translocation system and compared with their secretory capabilities in yeast. It has been shown that the conformation of the C-terminal region of the signal sequence and the length of the hydrophobic segment are important factors for efficient cleavage of the signal sequence.

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene.

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.

Alteration of N-terminal residues of mature human lysozyme affects its secretion in yeast and translocation into canine microsomal vesicles.

Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.

Conformational requirement of signal sequences functioning in yeast: circular dichroism and 1H nuclear magnetic resonance studies of synthetic peptides.

Recently, we have designed a series of simplified artificial signal sequences and have shown that a proline residue in the signal sequence plays an important role in the secretion of human lysozyme in yeast, presumably by altering the conformation of the signal sequence [Yamamoto, Y., Taniyama, Y., & Kikuchi, M. (1989) Biochemistry 28, 2728-2732]. To elucidate the conformational requirement of the signal sequence in more detail, functional and nonfunctional signal sequences connected to the N-terminal five residues of mature human lysozyme were chemically synthesized and their conformations in a lipophilic environment [aqueous trifluoroethanol (TFE) or sodium dodecyl sulfate micelles] analyzed by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. The helix content of the peptides, including functional (L8, CL10) and nonfunctional (L8PL, L8PG, L8PL2) signal sequences, was estimated from CD spectra to be 40-50% and 60-70%, respectively, indicating that the helical structure is more abundant in the nonfunctional signal sequences. Two-dimensional NMR analyses in 50% TFE/H2O revealed that each peptide adopted a helical conformation throughout the sequence except for a few residues at the N- and C-termini. Furthermore, H-D exchange experiments indicated that the helical structure of the C-terminal region of the functional signal sequences (L8 and CL10) was less stable than that of the nonfunctional signal sequences (L8PL and L8PL2). On the basis of these results, a model was developed in which the functional signal sequence is inserted in the membrane with a helical conformation and the C-terminal helix unraveled in an extended conformational form through an interaction with the signal peptidase.