[Geographic environment-related research advances on children's myopia: intraocular and environmental exposure factors and analytical methods].

Myopia has become a global phenomenon, transitioning into a significant public health issue of worldwide reach. The escalating prevalence of myopia may lead to an increase in the incidence of related complications, potentially resulting in irreversible vision damage for individuals. This not only causes considerable economic strain on societies but also poses a serious threat to vital sectors like national defense. This review outlines various external and internal exposure factors related to childhood myopia. It places particular focus on the analysis of the interaction between geographical environmental factors and internal exposure factors, and examines the limitations of applying traditional methods in studying the relationship between childhood myopia and geographical environmental factors. The paper also introduces two spatial regression methodologies based on frequency estimation and Bayesian estimation, summarizing their feasibility and merits when applied in the study of external exposure elements related to childhood myopia. Finally, it provides a fresh perspective on regional childhood myopia prevention strategies that are conscious of geographical environmental factors.

Aldose reductase inhibitors and nanodelivery of diabetic therapeutics.

Nanotechnology is a rapidly emerging drug-delivery system that makes possible the controlled release of small molecules, and nanodelivery of therapeutic molecules using nanoparticles or nanogels represents a major improvement for more focused delivery of such therapeutic molecules. The delivery of insulin for the control of diabetes mellitus (DM) and aldose reductase inhibitor (ARI) for diabetic complications may provide better treatment of diabetes. A structural overview of aldose reductase including computational docking experiments with HAR-1, various ARIs, aldose-keto reductase, and nanodelivery of insulin, ARI's, and drug molecules are described.

A synthetic triptycene bisquinone, which blocks nucleoside transport and induces DNA fragmentation, retains its cytotoxic efficacy in daunorubicin-resistant HL-60 cell lines.

In contrast to the parent triptycene (code name TT0), triptycene bisquinone (code name TT2) is cytostatic (IC50: 300 nM) and cytotoxic (IC50: 230 nM) in wild-type (WT), drug-sensitive HL-60 cells (HL-60-S) at day 4. Therefore, the effects of this new quinone antitumor drug were assessed and compared to those of daunorubicin (DAU, daunomycin) in the multidrug-resistant (MDR) HL-60-RV and HL-60-R8 sublines, which respectively overexpress P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP). In contrast to DAU, which loses its cytostatic [resistance factors (RFs): 22.9-35.7] and cytotoxic (RFs: 23.8-31.3) activities in MDR sublines, TT2 decreases tumor cell proliferation (RFs: 0.9-1.3) and viability (RFs: 0.9-1.5) as effectively in HL-60-S as in HL-60-RV and HL-60-R8 cells at days 2 and 4. Similarly, DAU inhibits the rate of DNA synthesis less effectively in MDR than in parental HL-60 cells (RFs: 8.1-11.9) but TT2 decreases the incorporation of 3[H]-thymidine into DNA to the same degree in HL-60-S, HL-60-RV and HL-60-R8 cells (RFs: 1.2). In contrast to DAU, which is inactive, the advantage of TT2 is its ability to block the cellular transport of purine and pyrimidine nucleosides in WT tumor cells, an effect which persists in both MDR sublines (RFs: 1.0-1.2). Moreover, the concentrations of DAU which induce maximal DNA cleavage in HL-60-S cells at 24 h lose all or most of their DNA-damaging activity in HL-60-RV and HL-60-R8 cells, whereas treatments with 4 microM TT2 produce similar peaks of DNA fragmentation in all WT and MDR cell lines. Since TT2 not only mimics the antitumor effects of DAU but also blocks nucleoside transport and retains its effectiveness in MDR cells that have already developed different mechanisms of resistance to DAU, this new quinone antitumor drug might be valuable to develop new means of polychemotherapy.

Synthetic 1,4-anthracenediones, which block nucleoside transport and induce DNA fragmentation, retain their cytotoxic efficacy in daunorubicin-resistant HL-60 cell lines.

Anthracene-1,4-dione and 6,7-dichloro-1,4-anthracenedione (code names AQ1 and AQ4, respectively) are cytostatic (IC50: 53 and 110 nM, respectively) and cytotoxic (IC50: 100 and 175 nM, respectively) in wild-type drug-sensitive HL-60-S tumor cells at day 4 in vitro. Therefore, the antitumor effects of these drugs were assessed and compared to those of daunorubicin (DAU) in HL-60-RV and HL-60-R8 tumor cells, which are, respectively, P-glycoprotein-positive and -negative multidrug-resistant (MDR) sublines. In contrast to DAU, which loses its cytostatic [resistance factors (RFs): 30.3-31.8] and cytotoxic (RFs: 48.8-58.1) activities in MDR sublines, AQ1 inhibits cell proliferation (RFs: 0.9-1.3) and cell viability (RFs: 1.4-1.6) as effectively in HL-60-RV and HL-60-R8 as in HL-60-S cells. Similarly, DAU decreases the rate of DNA synthesis less effectively in MDR sublines (RFs: 8.0-13.3) but AQ1 inhibits the incorporation of [3H]thymidine into DNA to the same degree in HL-60-S as in HL-60-RV and HL-60-R8 cells (RFs: 0.9-1.1). In contrast to DAU, which is ineffective, the advantage of AQ1 is its ability to block the cellular transport of purine and pyrimidine nucleosides in HL-60-S cells, an effect which persists in the MDR sublines (RFs: 1.1). AQ4, which mimics to a lesser degree all the antitumor effects of AQ1, except the inhibition of adenosine transport, also retains its effectiveness in MDR sublines (RFs: 1.1-3.1). The peaks of DNA cleavage caused by DAU and AQ1 in HL-60-S cells shift to lower concentrations with increasing times of drug exposure but DAU loses most of its ability to induce DNA fragmentation in MDR sublines, whereas the levels of AQ1-induced DNA cleavage at 16 and 24 h are nearly equivalent in HL-60-S, HL-60-RV and HL-60-R8 cells. Because they not only mimic the antitumor effects of DAU in the nM range but also block nucleoside transport and remain effective in tumor cells that have developed different mechanisms of MDR, AQ1 and AQ4 analogs might be valuable to develop new means of polychemotherapy.

1,4-Anthraquinone: an anticancer drug that blocks nucleoside transport, inhibits macromolecule synthesis, induces DNA fragmentation, and decreases the growth and viability of L1210 leukemic cells in the same nanomolar range as daunorubicin in vitro.

1,4-Anthraquinone (AQ) was synthesized and shown to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. In contrast, its dihydroxy-9,10anthraquinone precursor, quinizarin, was inactive. The antitumor activity of AQ was compared to that of daunorubicin (DAU), which is structurally different from AQ but also contains a quinone moiety. AQ is equipotent to DAU against L1210 tumor cell proliferation (IC50: 25 nM at day 2 and 9 nM at day 4) and viability (IC50: 100 nM at day 2 and 25 nM at day 4), suggesting that its cytostatic and cytotoxic activities are a combination of drug concentration and duration of drug exposure. Since AQ does not increase but rather decreases the mitotic index of L1210 cells at 24 h, it is not an antitubulin drug but might arrest early stages of cell cycle progression. Like DAU, a 1.5-3 h pretreatment with AQ is sufficient to inhibit the rates of DNA, RNA and protein syntheses (IC50: 2 microM) determined over 30-60 min periods of pulse-labeling in L1210 cells in vitro. In contrast to DAU, which is inactive, a 15 min pretreatment with AQ has the advantage of also inhibiting the cellular transport of both purine and pyrimidine nucleosides (IC50: 2.5 microM) over a 30 s period in vitro. Hence, AQ may prevent the incorporation [3H]thymidine into DNA because it rapidly blocks the uptake of these nucleosides by the tumor cells. After 24 h, AQ induces as much DNA cleavage as camptothecin and DAU, two anticancer drugs producing DNA strand breaks and known to, respectively, inhibit topoisomerase I and II activities. However, the concentration-dependent induction of DNA cleavage by AQ, which peaks at 1.6-4 microM and disappears at 10-25 microM, resembles that of DAU. The mechanism by which AQ induces DNA cleavage is inhibited by actinomycin D, cycloheximide and aurintricarboxylic acid, suggesting that AQ activates endonucleases and triggers apoptosis. The abilities of AQ to block nucleoside transport, inhibit DNA synthesis and induce DNA fragmentation are irreversible upon drug removal, suggesting that this compound may rapidly interact with various molecular targets in cell membranes and nuclei to disrupt the functions of nucleoside transporters and nucleic acids, and trigger long-lasting antitumor effects which persist after cessation of drug treatment. Because of its potency and dual effects on nucleoside transport and DNA cleavage, the use of bifunctional AQ with antileukemic activity in the nM range in vitro might provide a considerable advantage in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Triptycenes: a novel synthetic class of bifunctional anticancer drugs that inhibit nucleoside transport, induce DNA cleavage and decrease the viability of leukemic cells in the nanomolar range in vitro.

In contrast to their inactive parent compound triptycene (code name TT0), several triptycene (TT) analogs (code names TT1 to TT13), most of them new compounds, were synthesized and shown to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. The most potent rigid tetracyclic quinones synthesized so far are TT2 and its C2-brominated derivative, TT13. The antitumor activity of TT2 has been compared to that of daunomycin (DAU), a clinically valuable anthracycline antibiotic which is structurally different from TT2 but also contains a quinone moiety. TT2 inhibits the proliferation (IC50: 300 nM at day 2 and 150 nM at day 4) and viability (IC50: 250 nM at day 2 and 100 nM at day 4) of L1210 cells to the same maximal degree as DAU, suggesting that the cytostatic and cytotoxic activities of TT2 are a combination of drug concentration and duration of drug exposure. Since TT2 does not increase the mitotic index of L1210 cells at 24 h like vincristine, it is unlikely to be an antimitotic drug that disrupts microtubule dynamics. Like DAU, a 1.5-3 h pretreatment with TT2 is sufficient to inhibit the rates of DNA, RNA and protein syntheses determined over 30-60 min periods of pulse-labeling in L1210 cells in vitro (IC50: 6 microM). In contrast to DAU, which is inactive, a 15 min pretreatment with TT2 has the advantage of also inhibiting the cellular transport of nucleosides occuring over a 30 s period in vitro (IC50: 6 microM), suggesting that TT2 prevents the incorporation of [3H]thymidine into DNA because it rapidly blocks the uptake of [3H]thymidine by the tumor cells. After 24 h, TT2 induces as much DNA cleavage as camptothecin and DAU, two anti-cancer drugs producing DNA strand breaks and known to respectively inhibit DNA topoisomerase I and II activities. Interestingly, the abilities of TT2 to block nucleoside transport, inhibit DNA synthesis and induce DNA fragmentation are irreversible upon drug removal, suggesting that this compound may rapidly interact with various molecular targets in cell membranes and nuclei to disrupt the functions of nucleoside transporters and nucleic acids, and trigger long-lasting antitumor effects which persist after cessation of drug treatment. Because inhibition of nucleoside transport is highly unusual among DNA-damaging drugs, the use of bifunctional TTs with antileukemic activity in the nM range in vitro might provide a considerable advantage in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

6,7,8,9-Tetrahydro-3-methyl-1H-pyrano-[4,3-b]quinolin-1-one.

The condensation reaction of 4-amino-6-methyl-2-pyrone with 1-cyclohexenecarboxaldehyde and a catalytic amount of (S)-(+)-10-camphorsulfonic acid in toluene at 358 K gave a 1:2.5 ratio of the title compound, (1) (C13H13NO2), and 7,8,9,10-tetrahydro-1H-pyrano[4,3-c]isoquinoline-1-one, (2). The formation of (2) presumably proceeds through an intermediate imine. Both (1) and (2) show inhibitory activities against acetylcholinesterase and human aldose reductase. Of the three linear-fused rings of (1), both ring A and ring B are planar and the angle between these planes is 0.46 (13) degrees. While the two C atoms of cyclohexane ring C attached to its common atoms with ring B are in the plane of the latter, as expected, the remaining two C atoms of ring C are out of this plane, by 0.342 (4) and -0.402 (3) A, respectively.

Tricyclic pyrone analogs: a new synthetic class of bifunctional anticancer drugs that inhibit nucleoside transport, microtubule assembly, the viability of leukemic cells in vitro and the growth of solid tumors in vivo.

Tricyclic pyrones (TPs) may represent a novel synthetic class of microtubule (MT) de-stabilizing anticancer drugs previously shown by us to inhibit macromolecule synthesis, tubulin polymerization, and the proliferation of leukemic and mammary tumor cells in vitro. A linear skeleton with a N-containing aromatic ring attached at C3 of the top A-ring, a central pyran B-ring and a six-membered bottom C-ring with no alkylation at C7 are required for the antitumor activities of the lead compounds, a 3-pyridyl benzopyran (code name H10) and its somewhat weaker 2-pyridyl regioisomer (code name H19). Increasing concentrations of H10 do not alter the binding of [3H]vinblastine and [3H]GTP to tubulin but mimic the ability of unlabeled colchicine (CLC) to reduce the amount of [3H]CLC bound to tubulin, suggesting that TPs may interact with the CLC binding site to inhibit tubulin polymerization. Exogenous Mg2+ cations absolutely required for the binding of GTP to tubulin and MT assembly cannot overcome the antitubulin action of H10. H10 reduces the viability of L1210 cells in vitro (IC50: 0.5 microM) but its antitumor activity may be related to its ability to inhibit tubulin polymerization and rapidly increase the mitotic index rather than to induce DNA cleavage and apoptosis. The anticancer potential of TPs in vivo is demonstrated by the fact that i.p. injections of the water-soluble H10-HCl decrease the growth of solid tumors in mice inoculated s.c. with Lewis lung carcinoma. A critical finding is that the antimitotic H10 is a bifunctional anticancer drug, which also blocks the cellular transport of nucleosides (IC50: 6 microM) to inhibit DNA synthesis. Since few CLC site-binding antimitotic agents are active in solid tumor models in vivo, the ability of these new MT destabilizing TPs to totally block nucleoside transport might be valuable in polychemotherapy to arrest tumor cells at several phases of their cycle, potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Tricyclic pyrone analogs: a new class of microtubule-disrupting anticancer drugs effective against murine leukemia cells in vitro.

Novel 1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano [4,3-b][1]benzopyrans were synthesized in Hua's laboratory (code names H5, H10, H14 and H15) and tested for their ability to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. The aryl groups of these tricyclic pyrone (TP) analogs are either 3, 4-dimethoxyphenyl in H5 and H15 or 3-pyridyl in H10 and H14. Since 50 M H5 and H10 both inhibit DNA synthesis and tumor cell growth by 79-100%, concentrations 25 M were used in this study to assess the structure-activity relationships for this class of compounds. At 10-25 M, H5 and H14 are more potent inhibitors of DNA, RNA and protein synthesis than H10. In contrast, at 5-25 M, H10 is much more effective than H5 and H14 at inhibiting the growth of L1210 cells over a 4-day period. Interestingly, H15 inhibits DNA synthesis as much as H10 but fails to alter tumor cell growth. This discrepancy between the ability of TPs to inhibit macromolecule synthesis and leukemic cell growth suggests that other molecular targets may be involved in the antitumor action of these drugs. Their short-term inhibition of nucleic acid synthesis is reversible following drug removal but their long-term inhibition of tumor cell growth is not. Moreover, 25 M H5 and H10 are not cytotoxic at 2 days but equally decrease cell viability at 4 days, suggesting that the potent and irreversible inhibition of cell proliferation observed 1-4 days after H10 treatment is not solely caused by drug cytotoxicity. The effectiveness of H10 as inhibitor of L1210 cell growth is comparable to that of a spectrum of representative anticancer drugs. A critical finding is that 5 M H10 blocks the polymerization of purified tubulin by 90% and, therefore, may be a novel microtubule de-stabilizing drug. Indeed, H10 inhibits tubulin polymerization and L1210 cell growth as much as 5 M of vincristine (VCR). In contrast, 5 M H5 alters neither tubulin polymerization nor tumor cell growth. The ability of H10 to disrupt microtubule dynamics indirectly suggests that TPs may be novel cell cycle-specific anticancer drugs useful for arresting mammalian cells in mitosis.

Antitumor activity of tricyclic pyrone analogs, a new synthetic class of microtubule de-stabilizing agents, in the murine EMT-6 mammary tumor cell line in vitro.

Novel tricyclic pyrone (TP) analogs synthesized in Hua's laboratory (code names H10, H14 and H16) were tested against a spectrum of known antimitotic drugs for their ability to disrupt microtubule (MT) dynamics, alter the mitotic index, and prevent murine EMT-6 mammary sarcoma cells from synthesizing DNA and proliferating in vitro. At 2-10 microM, H10 inhibits DNA synthesis, tubulin polymerization and tumor cell growth to a greater degree than H14, whereas H16 has no effect. A linear skeleton with a pyridyl ring at C-3 of the A-ring, a pyran B-ring and no alkylation at C-7 of the C-ring is required for the antitumor activity of these TPs. Since H10 mimics the effect of vincristine (VCR), but not that of paclitaxel, on tubulin polymerization, TPs may represent a novel synthetic class of MT de-stabilizing anticancer drugs. H10 is less potent than VCR against tubulin polymerization (IC50: 1.5 microM versus 0.15 microM) and tumor cell proliferation (IC50: 1.5 microM versus 5 nM) but inhibits DNA synthesis (IC50: 10 microM) more effectively than all other MT-disrupting agents tested, except tubulozole-C. Although TPs disrupt DNA synthesis and might affect several phases of the cell cycle, the ability of H10 to increase the percentage of mitotic cells indicates that these novel compounds may be cell cycle-specific anticancer drugs useful for arresting mammalian cells in M-phase.

Anticancer activities of 2,5,8,9-substituted 6-oxo-1,2,3,4,5,6-hexahydrophenanthridines on multi-drug-resistant phenotype cells.

Lycorine is a member of the alkaloid group from the bulb of the amaryllidaceae. This drug has been reported to have anticancer activity. Several synthetic intermediates obtained during the synthetic study of anticancer drugs based on the lycorine structure, were tested for anticancer activity using three cell lines: L1210 and HL60 cell lines which were resistant (R) or sensitive (S) to adriamycin. The two synthetic intermediates, 2-acetoxy- and 2-hydroxy-5-allyl-8,9-methylenedioxy-6-oxo-1,2,3,4,5,6-hexahydr ophenanthridine (1 and 2), both had anticancer activity in all three cell lines. However, the LD50 for the precursors was about 20 fold greater than for the native lycorine. Both 1 and 2 were cytotoxic to the adriamycin-resistant cell line, indicating that these drugs are not affected by the multidrug resistance factors. When low doses of the compounds were used, the HL60R cell line could be induced to differentiate to a cell which expressed a macrophage specific protein. These results suggest that phenanthridines 1 and 2 can be used on cells which are resistant to adriamycin, and that one mechanism of action is the induction of differentiation.

Antitumor activity of novel tricyclic pyrone analogs in murine leukemia cells in vitro.

New tricyclic pyrone derivatives were synthesized and tested for their ability to prevent L1210 leukemic cells from synthesizing DNA and growing in vitro. At 50 microM, a pyripyropene analog has no effect, whereas four pentahydro-3-aryl-1-oxopyrano[4,3-b][1]benzopyrans all inhibit DNA synthesis by 79-91% and tumor cell growth by 93-100%. These inhibitory effects are concentration dependent with IC50 around 8.5 microM for DNA synthesis at 2 hours and 1.1 microM for tumor cell growth at 4 days. The aryl groups of these antitumor agents are either 3,4-dimethoxyphenyl or 3-pyridyl. Introduction of a methyl group at C5a and a formyloxy or hydroxy group at C6 does not alter the antitumor effects of the 3,4-dimethoxyphenyl benzopyrans but reduces those of the 3-pyridyl benzopyrans, which, at 50 microM, inhibit DNA synthesis by only 32-49% and fail to alter tumor cell growth. The 4-hydroxy-6-(3-pyridyl)-2-pyrone has no effect and the tricyclic pyrones lacking aryl groups have very little inhibitory effects on DNA synthesis, suggesting that a greater conjugation is required for the antitumor activity. These molecules have never been reported and might be valuable to develop a new class of anticancer drugs.

Characterization of the antitumor-promoting activity of camptothecin in SENCAR mouse skin.

(+)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S phase cells, was tested topically for its ability to inhibit skin tumor initiation by 7,12-dimethylbenz[a]anthracene (DMBA) and complete tumor promotion by 12-0-tetradecanoylphorbol-13-acetate (TPA) in SENCAR mice. Even though CPT does not prevent the covalent binding of a subcarcinogenic dose of DMBA to DNA, it enhances early inhibition of DNA synthesis caused by this initiator and may decrease the essential role of DNA replication in tumor initiation. Indeed, CPT (400 nmol) applied 4 h before or 1 h after DMBA inhibits the yield, but not the incidence, of skin tumors initiated by this compound. Moreover, because it inhibits TPA-stimulated DNA synthesis at 16 h when applied 12 h after the tumor promoter, CPT partially decreases tumor initiation when DMBA is applied 16 h after a TPA pretreatment. CPT (400 nmol) applied 1 h before or 4, 12, 24 or 48 h after each promotion treatment with TPA remarkably inhibits the incidence and yield of skin tumors promoted by this agent. CPT delays and inhibits promotion of skin tumors the most when applied 12-24 h after each TPA treatment, at times when it can block the stimulation of DNA synthesis that follows the period of early inhibition caused by TPA. The ability of post-treatments with 25, 100 and 400 nmol CPT to inhibit skin tumor promotion is dose dependent. In the TPA (stage I)-mezerein (stage 2) protocol CPT (400 nmol) post-treatment inhibits both the first and second stages of tumor promotion, related to its ability to decrease the DNA and ornithine decarboxylase responses required for stages 1 and 2 respectively. The classic model of multistage skin carcinogenesis, therefore, may be valuable to determine if novel CPT analogs are more effective than their parent compound at inhibiting tumor initiation, promotion and progression.

Camptothecin post-treatments inhibit the biochemical events linked to the tumor-promoting component of carcinogenesis in mouse epidermis in vivo.

20(S)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S-phase cells, was tested topically for its ability to inhibit the biochemical markers of skin tumor promotion. CPT has no or very little inhibitory effect on the covalent binding of an initiating dose of 7,12-dimethylbenz-[a]anthracene (DMBA) to DNA at 24 hr, but CPT post-treatments remarkably inhibit stimulations of DNA synthesis caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 16 hr and a carcinogenic dose of DMBA at 7 days. CPT is a much more potent inhibitor if it is applied 10-14 hr after TPA or 4-6 days after DMBA, when DNA synthesis starts being stimulated after the periods of early inhibition caused by TPA and DMBA. When applied 12 hr after the tumor promoter, the ability of 3-3,000 nmol of CPT to inhibit TPA-stimulated DNA synthesis at 16 hr is dose-dependent. A single dose of 500 nmol of CPT inhibits the entire time course for the stimulation of DNA synthesis observed 16-64 hr after TPA. CPT also reduces the various DNA responses to chronic TPA treatments and structurally different non-TPA-type tumor promoters. CPT may indirectly decrease the ornithine decarboxylase-inducing activity of multiple TPA treatments because it can inhibit the stimulation of RNA synthesis by this compound. However, CPT fails to alter TPA-stimulated hydroperoxide production in relation to its inability to inhibit TPA-stimulated protein synthesis. On an equal dose basis, topotecan and 10-hydroxycamptothecin are more and less effective than CPT, respectively, whereas 10,11-methylenedioxycamptothecin is much more potent than its parent compound at inhibiting the DNA response to TPA. A single dose of 400 nmol of CPT has no effect on tumor initiation when applied 4 hr before or 1 hr after a single subcarcinogenic dose of DMBA. In contrast, 400 nmol of CPT chronically applied 1 hr before or 24 hr after each treatment with TPA remarkably inhibits the complete tumor-promoting activity of this agent. CPT post-treatments also inhibit the respective activities of TPA and mezerein in the 1st and 2nd stages of skin tumor promotion.

Detection of oxidation products in individual neurons by fluorescence microscopy.

In the study of the central nervous system, it is necessary to address mechanisms by which cells are injured. In vitro investigations using cells in culture allow sharply focused mechanistic questions to be addressed; however, these studies have often been limited by the sensitivity constraints of assays. Many assays for oxidative products require large amounts of cells that must be disrupted. The extent of oxidation in individual cells is therefore unknown and the results yield an average among different cell types. This is inconvenient in cultures of the nervous system which often have multiple cell types. Using a newly developed method for visualizing oxidation products in individual cells, we have examined oxidation in neurons in culture. The method uses a hydrazide, biotin-4-amidobenzoic hydrazide, to bind carbonyls generated from oxidation. Biotin is detected by streptavidin conjugated with a fluorescent dye. Neurons in culture were exposed to 0.1 to 100 microM ferrous sulfate and fluorescence was visualized and quantitated using confocal laser microscopy. Low levels of oxidation (0.1 microM ferrous sulfate) were easily detected with this method. Iron concentration and fluorescence intensity correlated highly (r = 0.991). As an indicator of the sensitivity of this new method, carbonyl content in the cultures was also quantitated using the 2,4-dinitrophenylhydrazine assay (DNPH). The DNPH assay failed to detect the low levels of oxidation which were detected by the biotin-4-amidobenzoic hydrazide method. Fluorescence intensity partially paralleled loss of neuronal viability. Low concentrations of iron (0.1 and 1.0 microM) did not produce significant neuronal death; however, higher concentrations (10 and 100 microM) produced 19 and 53% neuronal loss, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

(4aR,8R,SR)-8-(p-tolylsulfinyl)-2,3,4,4a,5,6,7,8-octahydro-4a-quinolinol.

The title compound, C16H21NO2S, isolated from the reaction of lithiated racemic 2,3,4,4a,5,6,7,8-octahydroquinoline with 0.5 equivalents of (-)-(1R,2S,5R)-menthyl (SS)-p-toluenesulfinate, possesses the unusual conformation in which the OH and bulky p-toluenesulfinyl groups are in axial positions. The S=O bond distance of 1.519 (3) A is longer than expected because of intramolecular hydrogen bonding between the hydroxyl group and the sulfinyl O atom.

Ethyl (2R,3R,SR)-3-(ethoxycarbonyloxy)-2-[1-(p-tolylsulfinyl) cyclopropyl]-1-pyrrolidinecarboxylate.

The title compound, derived in four steps beginning with the cyclopropanation reaction of the anion of the alpha-sulfinyl ketimine (4R,SS)-4-(tert-butyldimethylsilyloxy)-3,4-dihydro-5-(p-tolysul finyl)methyl)-2H-pyrrole with 2-chloroethyl trifluoromethanesulfonate, contains a shorter than expected bond (C(7)--C(8) 1.48(l) A) and a larger than expected angle (S(1)--C(6)--C(2) 123.2(6) in the cyclopropane framework.

Structure of sp-9-hydroxy-9-pivaloylfluorene, product of base-catalyzed autoxidation of ap-9-pivaloylfluorene.

1-(9-Hydroxyfluoren-9-yl)-2,2-dimethyl-1-propanone, C18H18O2, M(r) = 266.34, orthorhombic, Pbcn, a = 18.917 (10), b = 11.843 (8), c = 13.177 (7) A, V = 2952 (5) A3, Z = 8, Dx = 1.198 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.72 cm-1, F(000) = 1136, T = 296 K, R = 0.044 for 1042 unique observed reflections. Conversion of ap-9-pivaloylfluorene, ap-(I), into lithiated (I)-9-anion followed by the addition of MeOH, then H2O, led to unexpected hydroxylation to provide 20-40% of sp-9-hydroxy-9-pivaloyfluorene, sp-(II), and 60-80% recovery of ap-(I). The singular sp conformation of (II) in solution suggested by NMR was confirmed in the crystalline state by X-ray diffraction which showed the O(1)-C(9)-C(10)-O(2) torsion angle approximately 0 degrees and the C(10) = O(2)...H(9)-O(1) non-bonding distance 1.95 (4) A, suggesting strong intramolecular hydrogen bonding in this conformation.

Structure of cis,cis-4,6-diphenyl-2-(2-propenyl)-1,3-dioxa-2-phosphorinane 2-oxide.

C18H19O3P, M(r) = 314.32, monoclinic, P2(1)/n, a = 5.726 (6), b = 14.234 (7), c = 20.08 (1) A, beta = 92.54 (7) degrees, V = 1635 (4) A3, Z = 4, Dx = 1.277 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.71 cm-1, F(000) = 664, T = 296 K, R = 0.042, 1813 unique observed reflections. The X-ray structure determination of the title compound shows that the dioxaphosphorinane ring has a chair conformation in which the phosphoryl O atom (P=O) is equatorial, which explains the absence of substantial NMR deshielding by its P=O group on H(4) and H(6), which are axial.