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A letter to the editor is presented in response to the Fall 2006 issue of "Gems & Gemology."

A letter to the editor is presented in response to the article on the characterization of Malossi hydrothermal synthetic emerald.

Pharmaceutical companies develop specialized therapies to treat late stage cancer. In order to accelerate life-saving treatments and reduce animal testing, compounds to treat life-threatening malignancies are allowed modified requirements for preclinical toxicology testing. Limited data packages in early drug development can present product quality challenges at multi-product manufacturing facilities. The present analysis established an endpoint-specific threshold of toxicological concern (TTC) for developmental and reproductive toxicity (DART) for anticancer compounds. A comprehensive database was created consisting of over 300 no-observed adverse effect levels (NOAELs) for DART of 108 anticancer compounds. The 5th percentile NOAEL for developmental and reproductive toxicity was 0.005 mg/kg/day (300 μg/day), resulting in a human exposure threshold of 3 μg/day assuming standard uncertainty factors and a 60 kg human bodyweight. The analysis shows this threshold is protective for developmental and reproductive toxicity of highly potent groups of anticancer compounds. There were similar TTC values calculated for direct-acting and indirect-acting anticancer compounds., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Expression of an estrogen-regulated reporter gene, growth of MCF-7 cells in the presence of 17beta-estradiol (E2) or E2 plus TCDD, and DNA microarray plus real time quantitative PCR analyses of gene expression in MCF-7 cells were used to evaluate the effects of TCDD, a known E2 antagonist, on E2-regulated gene expression in human cells. TCDD added simultaneously with E2 exhibited significantly decreased E2-associated upregulation of reporter gene expression compared with cells treated with E2 alone, and decreased E2 enhancement of mitosis in MCF-7 cells. MCF-7 cells treated with E2 or E2 plus TCDD and DNA microarray-evaluated to determine patterns of gene expression, showed substantial differences in gene expression in TCDD-treated cells compared with E2-treated cells. Of the 2400 genes on the Perkin Elmer global array microchip utilized for this analysis, a minimum of 317 were significantly upregulated and 488 were significantly downregulated. Of these, the gene encoding insulin receptor substrate-1 (IRS-1), the protein product of which has been previously reported to be decreased, missing, altered, or defective in persons with type 2 diabetes mellitus, was evaluated by real time quantitative PCR to corroborate the array data. An evaluation of the potential consequences of TCDD-altered IRS-1 downregulation is presented.

Halogenated aromatic hydrocarbons (HAHs) such as dibenzo-p-dioxins are known to alter cognitive function. However, the cellular basis of this disruption is not well understood. One possible deleterious effect of exposure to HAHs could be on gap junctional intercellular communication (GJIC) between neurons and astroglia in the brain. As such, this study examined the effects of the highly toxic prototypic HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on GJIC in rat hippocampal primary cell culture. Initial measurements of fluorescence recovery after photobleaching (gap-FRAP) showed dye transfer between astroglia and neurons. N-octanol, a lipophilic alcohol known to uncouple cells by decreasing the open probability of gap junctional channels blocked astroglial-neuronal (A-N) communication as well as astroglial-astroglial (A-A) communication. TCDD initially downregulated GJIC between neurons and astroglia of treatment, but had no effect on astroglial cell pairs. These results indicate the presence of GJIC between neurons and astroglia in culture and demonstrate different sensitivities of gap junction responses to TCDD in homologous and heterologous cell pairs. The finding that 2,3,7,8-TCDD disrupts GJIC through A-N but not A-A channels may have important implications for impaired brain function resulting from developmental exposure to TCDD.

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.

This paper reports the results from in vitro experiments utilizing vital fluorescent probes and biochemical assays to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and related compounds in primary rat astroglia in an effort to identify the cellular site(s) involved in toxicity. Application of 100 nM 2,3,7,8-TCDD, a strong Ah receptor agonist, resulted in altered astroglial intracellular Ca2+, a significant decrease in glutathione, a disrupted mitochondrial membrane potential, a significant decrease in glutamine synthetase immunoreactivity and eventual loss of pH maintenance. In contrast, application of 10 microM 1,2,3,4-TCDD, a weak Ah receptor agonist, had no effect on any parameters measured. These findings, coupled with the identification of the 9-10S cytosolic Ah receptor in cultured rat astroglia, are consistent with typical structure-activity relationships observed for other Ah receptor mediated responses. However, the time course of the Ca2+, as well as other responses observed in this study, suggest that the above effects may not necessarily involved the formation of the nuclear Ah receptor complex.

This study examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and related compounds on the uptake of intracellular calcium ([Ca2+]i) in primary cultures of rat hippocampal neuronal cells. [Ca2+]i levels were detected and quantified by interactive laser cytometry with microscopic image analysis. Cells were noninvasively labeled with fluo-3/AM and all experiments were conducted on cultured rat hippocampal neurons 14 days in culture. Treatment of cell cultures with 2,3,7,8-TCDD (10-100 nM) resulted in a rapid concentration-dependent increase in [Ca2+]i associated with a decrease in mitochondrial membrane potential and activation of alpha-protein kinase C (alpha-PKC). In contrast, 1,2,3,4-TCDD, a weak Ah receptor agonist, had no effect on [Ca2+]i at concentrations as high as 10 microM and similar results were also observed for 2,2',5,5'-tetrachlorobiphenyl. Maximal [Ca2+]i was observed within 30 s after addition of 2,3,7,8-TCDD and remained elevated (at higher concentrations) above resting levels for the duration of the experiment. This rapid increase in [Ca2+]i was blocked by addition of EDTA (2 mM) to the external medium or by pretreatment of the cells with the calcium channel antagonist nifedipine (10 microM). However, pretreatment of the cells with 100 microM cycloheximide failed to block calcium uptake in neuronal cells. These data indicate that rat hippocampal neuronal cells are responsive to 2,3,7,8-TCDD; however, the mechanism is not associated with altered gene transcription and may involve cellular targets.

The results of diverse in vitro neurotoxicity studies demonstrate that there are variations in cellular responsiveness between different types of neural cells. In contrast to experimental systems that have reported cellular responses to relatively high concentrations of various PCB congeners, our studies with rat hippocampal neural cells indicate that neurons and astroglia are responsive to relatively low levels of TCDD. However, these responses are probably not mediated through the classical Ah receptor pathway, which involves nuclear Ah receptor-mediated modulation of gene expression. It has recently been reported that TCDD-induced phosphorylation and other responses can be observed in some cell lines within minutes after treatment (20), and that cell membrane or cytosolic receptors may also play a role in mediating these effects. Future studies are required to determine both Ah receptor-dependent and -independent pathways associated with the neurotoxicity of PCBs, TCDD, and related compounds. The report that low-level dietary or background exposure to HAHs (32) results in neurobehavioral deficits is still a perplexing problem also requiring additional research and consideration of other dietary factors that may contribute to these effects. For example, we have recently been comparing the toxic effects and relative potencies of TCDD (exodioxins) and other "natural occurring" compounds (endodioxins) such as indole-3-carbinol (vegetables) and polynuclear aromatic hydrocarbons (PAHs, cooked foods), which also bind to the Ah receptor. Dr. Clynn Wilker has shown that in utero exposure of rats to indole-3-carbinol and chrysene (a PAH) cause demasculinization of the adult offspring as previously reported for TCDD (19). Thus, the neurotoxicity of low-level dietary exposure to HAHs should at least consider other possible confounding factors, including dietary endodioxins.

Melatonin, a hormone produced in the pineal gland and released into the general circulation on a diurnal basis, has been implicated in many behavioral processes, where it has been shown to have anxiolytic, sedative, and anticonvulsant effects. Male gerbils (Meriones unguiculatus) injected daily with melatonin (25 micrograms, s.c.) exhibited a reduced seizure response to pentylenetetrazol (PTZ, 60 mg/kg, s.c.). The present studies determined 1) whether melatonin's effect was related to the time of day that it was administered and 2) whether a single acute injection of melatonin at various doses could produce anticonvulsant activity. Gerbils provided with 13 weeks of daily melatonin injections (25 micrograms, s.c.) exhibited fewer convulsions after PTZ treatment irrespective of the time of day melatonin was injected. In addition, the melatonin-treated gerbils had lower mortality rates (1/12) than the untreated or vehicle-injected gerbils (5/12). On the other hand, single acute injections of melatonin (0.1-10 mg/kg, i.p.) produced no anticonvulsant activity. It appears that the anticonvulsant effects of melatonin occur only after the animals are chronically exposed to the indole. In addition, melatonin's anticonvulsant ability may utilize a different mechanism than those involved in its endocrine effects, since no diurnal difference in melatonin's anticonvulsant activity was observed.

A new procedure for the measurement of gamma-aminobutyric acid, norepinephrine, dopamine, serotonin and 5-hydroxyindoleacetic acid from the same brain region was developed. In general, two separate high-performance liquid chromatographic runs were performed, one for the gamma-aminobutyric acid determination and one for the determination of the monoamines. The electrochemical detection of gamma-aminobutyric acid was determined by a new procedure that utilized a small aliquot of the brain sample prepared for monoamine measurement. This assay was linear and parallel between 6 and 200 ng per 20-microliters injection with 5-aminovaleric acid utilized as an internal standard. Inter-assay variability averaged 5% throughout the assay with gamma-aminobutyric acid values in the gerbil hypothalamus of 344 micrograms/g. The catecholamine assay has been characterized previously and utilizes 3,4-dihydroxybenzylamine as an internal standard with less than 5% variability. Norepinephrine, dopamine, serotonin and 5-hydroxyindoleacetic acid levels in the gerbil hypothalamus averaged 2922, 729, 797 and 272 ng/g, respectively. This new protocol allows a wide range of neurochemicals to be determined and evaluated from the same brain region.

Autoradiography of colony replicas immobilized on filter paper was used to isolate a Chinese hamster ovary cell line deficient in incorporation of radiolabeled fucose into a trichloroacetic acid-insoluble fraction. This cell line, called 62.1, has the same growth rate at 37 degrees C as wild-type cells, but incorporates five times less fucose into acid-insoluble radioactivity. Chemical analysis of fucose bound to macromolecules also showed a fivefold reduction in the mutant. The fucoproteins of the mutant cell line differ qualitatively from those of wild-type cells as visualized by SDS gel electrophoresis fluorography; no differences were detected between total proteins as visualized by coomassie blue staining. The macromolecular sialic acid content of the mutant was somewhat higher than the wild type (20%). Studies of the synthesis of the glycoprotein of vesicular stomatitis virus in mutant and wild-type cells showed that the mutant is unable to synthesize complex-type N-linked oligosaccharides. Enzyme assays show that ths defect in the mutant is due to reduction in UDP-N-acetylglucosamine-glycoprotein N-acetyl-glucosaminyltransferase, a key enzyme in the assembly of complex glycopeptides. Hybridization studies have shown that mutant 62.1 has common mutations belonging to the same complementation group as mutant PhaR1-1. This latter mutant was previously isolated using lectin resistance by Stanley et al. (1975) and was also deficient in the above N-acetyl-glucosaminyltransferase.