Andrew Z. Mason

The metabolism of testosterone by the periwinkle (Littorina littorea) in vitro and in vivo: Effects of tributyl tin

Abstract Exposure to tributyl tin (TBT) at concentrations as low as 0.5 ng/liter has been associated with disrupted sexual physiology (imposex) in 40–50 species of marine gastropod. Imposex is a state of pseudohemaphrodism in which females exhibit nonfunctional secondary male characteristics. It has been suggested that the mechanism underlying imposex involves disrupted metabolism of endogenous sex hormones and in particular inhibition of cytochrome P450-dependent aromatization of androgens to estrogens. In the current study, the effects of TBT on the ability of the Periwinkle ( Littorina littorea ) to metabolize the androgenic sex steroid testosterone was examined in vitro and in vivo . Microsomes were prepared from ‘digestive gland’ (visceral complex including the gonads) and combined kidney and gill fractions. CO-ligated reduced difference spectra contained peaks at 459 nm and calculated P450 contents of 0.3 and 0.05 nmol/mg. Digestive gland microsomes were found to be capable of oxidative metabolism of testosterone in either the presence or absence of NADPH. In the absence of NADPH, testosterone was metabolized to androstenedione, 6β-, 6α- and 15β-hydroxytestosterone, 17β-estradiol and an unidentified metabolite possibly 3α-androstene-17β-diol. In the presence of NADPH the same products were produced at a higher rate but the major products formed were the products of the NADPH-dependent steroid 5α-reductase-3α(β)hydroxysteroid dehydrogenase pathway; dihydrotestosterone (DHT) and 3α(β)androstane-17β-diols (DHT diols). TBT, even at high concentrations up to 100 μM, had only modest effect on P450-dependent testosterone metabolism in vitro , producing increases in androstenedione formation and only 30–40% inhibition of aromatase. In vivo , [ 14 C] testosterone was injected directly into the cephalopedal sinus of adult snails and animals maintained in sea water at 15 °C for 42 h in the presence of 0, 0.5 and 5 mM TBT. Testosterone was almost completely metabolized during this time, predominantly to water-soluble sulfur conjugates of testosterone, the 5α-reduced products and 6α-hydroxytestosterone. However, with increasing concentrations of TBT, more radioactivity was retained within the animal and was increasingly associated with organic extractable unmetabolized testosterone and its phase I products androstenedione, dihydroandrostenedione (DHA), DHT and DHT-diols. Thus it appears that, in vivo , TBT inhibits sulfur conjugation of testosterone and its phase I metabolites and their excretion resulting in a build-up of pharmacologically active androgens in the tissues. This data are consistent with the hypothesis that TBT-induced imposex in sensitive gastropods, such as stenoglossans, may arise from peturbations in sex steroid metabolism. However, the major biochemical targets of TBT appear to be steroid conjugation and excretory transport mechanisms rather than P450-dependent oxidative pathways such as aromatase.

Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

Relationships between subcellular distributions of cadmium and perturbations in reproduction in the polychaete Neanthes arenaceodentata

Abstract The accumulation and subcellular distribution of Cd in the polychaete worm, Neanthes arenaceodentata , were examined following an eleven-week period of exposure to a range of free cadmium ion activities, [Cd 2+ ]. The accumulation of Cd in N. arenaceodentata was directly proportional to [Cd 2+ ] in seawater at lower concentrations (10 −12 M to 10 −10 M) but deviated from proportionality at higher concentrations (10 −9 M and 10 −8 M). This deviation in proportionality at higher [Cd 2+ ] was attributable to a relative increase in the concentration of metal associated with the metallothionein and the very low molecular weight metal-ligand pools of the cytosol. Reproductive potential was also monitored in these organisms to examine the ecological significance of Cd accumulation and shifts in its subcellular distribution. Perturbations in reproduction were observed at 10 −8 M [Cd 2+ ] which coincided with the deviation in proportionality of Cd accumulation and increased accumulation of Cd in the cytosol.

Heavy Metal in Children’s Tooth Enamel: Related to Autism and Disruptive Behaviors?

To examine possible links between neurotoxicant exposure and neuropsychological disorders and child behavior, relative concentrations of lead, mercury, and manganese were examined in prenatal and postnatal enamel regions of deciduous teeth from children with Autism Spectrum Disorders (ASDs), high levels of disruptive behavior (HDB), and typically developing (TD) children. Using laser ablation inductively coupled plasma mass spectrometry, we found no significant differences in levels of these neurotoxicants for children with ASDs compared with TD children, but there was marginal significance indicating that children with ASDs have lower manganese levels. No significant differences emerged between children with HDB and TD children. The current findings challenge the notion that perinatal heavy metal exposure is a major contributor to the development of ASDs and HDB.

Copper economy in Chlamydomonas: Prioritized allocation and reallocation of copper to respiration vs. photosynthesis

Significance Many inorganic elements, because they are required for catalysis or as structural components in biomolecules, are essential nutrients for life. In a situation of poor nutrition, organisms economize on the use of these elements by choosing alternate chemistries to achieve the same function. The mechanism of copper economy in Chlamydomonas involves replacement of copper-containing plastocyanin with heme-containing cytochrome (Cyt) c6. The copper that is saved by this substitution is used instead for Cyt oxidase biosynthesis. Copper recycling requires proteolysis of plastocyanin dependent on a copper-sensing transcription factor. A candidate protease has been identified. Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

Elemental mapping in fossil tooth root section of Ursus arctos by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to map the matrix (Ca, P) and trace (Ba, Sr, Zn) elements in the root section of a fossilized brown bear (Ursus arctos) tooth. Multielemental analysis was performed on a (2.5 × 1.5)cm(2) area. For elemental distribution, a UP 213 laser ablation system was coupled either with a quadrupole or a time of flight ICP-MS. The cementum and dentine on the slice of the sample surface were clearly distinguishable, especially changes in elemental distribution in the summer and winter bands in the fossil root dentine. Migration and diet of U. arctos were determined on the basis of fluctuations in Sr/Zn ratio and their contents. Quantification was accomplished with standard reference material of bone meal (NIST 1486) and by the use of electron microprobe analysis (EMPA). Changes in Sr/Zn and Sr/Ba ratios relating to the season, and composition of food during the lifetime of the animal are discussed on basis of analysis of light stable isotopes. It was observed that there was an increase in the Sr/Zn ratio during the winter season caused by a reduction of food intake during hibernation. Above mentioned inferences drawn from elemental data obtained by LA-ICP-MS were confirmed independently by determination of carbon, nitrogen and strontium isotopes. Moreover, diagenesis and its interfering influence on the biogenic composition of cementum and dentine were resolved. According to the distribution and/or content of the element of interest, post-mortem alterations were revealed. Namely, U, Na, Fe, Mg and F predicate about the suitability of the selected area for determination of migration and diet.

CONCENTRATION OF CONTAMINANTS IN BREEDING BIRD EGGS FROM THE COLORADO RIVER DELTA, MEXICO

Organic contaminants (organochlorine [OC], organophosphorus [OP] pesticides and polychlorinated biphenyls [PCBs]), and metals (As, Cd, Hg, Pb, and Se) are a concern to avian health in the Colorado River delta, Mexico. We determined concentrations of contaminants in eggs of three breeding species of birds from the delta (mourning doves [Zenaida macroura], burrowing owls [Athene cunicularia], and marsh wrens [Cistothorus palustris]). We collected 27 eggs of mourning doves, eight eggs of burrowing owls, and 18 eggs of marsh wrens for analyses. Polychlorinated biphenyls, OC, and OP pesticides were analyzed by gas chromatography equipped with an electron capture detector, and metals were analyzed by inductively coupled plasma mass spectrometry. The non-ortho PCB congeners (PCB 77 and 126) were found in mourning dove and burrowing owl eggs at concentrations in which hatchability can be affected. Mean selenium concentration found in marsh wren eggs (5.6 microg/g dry wt) exceeded the level of concern. Arsenic and Cd were found at higher than normal concentrations, Hg concentrations did not exceed the level of concern in any of the species, and Pb concentrations were higher in eggs of species subject to hunting. With the exception of lead, marsh wren eggs contained the highest metal concentrations.

A study of Cu turnover in proteins of the visceral complex of Littorina littorea by stable isotopic analysis using coupled HPLC-ICP-MS.

A two-dimensional HPLC system, tandemly coupled to an ICP-MS, has been used to study copper accumulation and turnover in the visceral complex cytosol of the gastropod, Littorina littorea. Animals were exposed for 8 weeks to NTA-buffered seawater containing stable isotopic 65Cu and then transferred to media containing stable isotopic 63Cu. The free ion activity of each isotope was maintained at 10(-11) M. Size exclusion (SE) HPLC showed Cu associated with haemocyanin (HC) and metallothionein-like (MT) proteins in two ligand pools with apparent molecular weights of >300 kDa and approximately 17 kDa, respectively. The MT pool was inducible by Cu, could assimilate the metal from both intrinsic and extrinsic sources and showed a higher rate of Cu accumulation and turnover than the HC pool. The induction of this pool also caused the sequestration and cytosolic redistribution of Zn, Cd, Pb, Mn and Co. Further fractionation of the MT pool by ion-exchange (IE) HPLC revealed that the Cu was associated with a single, major isoform of the protein that was Cu inducible and also bound trace quantities of Zn and Pb. A number of additional metal containing proteins were also resolved by IE. the most prominent of which also bound Pb, Mn and minor quantities of Zn. The significance of these findings in metal homeostasis and detoxification is discussed.

Cytosolic redistribution and enhanced accumulation of Cu in gill tissue of Littorina littorea as a result of Cd exposure

Abstract Metallothionein (MT) binds Cd and Cu with differential affinity. The stability constant of Cu-thioneine is approximately two orders of magnitude greater than that of Cd-thioneine. Thus, it may be expected that MT, induced by Cd exposure, may sequester cytosolic Cu in preference to Cd, resulting in a concomitant redistribution of the metal. In this study, two groups of the marine gastropod, Littorina littorea, were exposed to NTA-buffered seawater solutions, which contained equal free-ion activities of Cu but either low or high Cd free-ion activities. ICP-MS analyses of gill-tissue cytosols show an increase in the concentrations of both Cd and Cu upon exposure to high concentrations of Cd. SE-HPLC profiles indicate that Cd is accumulated in a Cd and Cu metal-binding-ligand pool that may contain MT. Furthermore, a redistribution of Cu to this pool from two low-molecular-weight ligand pools is evident. If these results are reflective of the in-vivo situation and Cu is being sequestered from essential Cu-containing enzymes, then MT induction by Cd intoxication may ultimately result in metal-induced toxicity brought about by cellular Cu deficiency.

The effect of estrogen on cadmium distribution in rainbow trout (Oncorhynchus mykiss)

Abstract The effects of injections of 17β-estradiol (E2) and Cd, on the distribution of Cd and the induction of metallothionein (MT) mRNA and vitellogenin mRNA was investigated. Bone and liver were the main organs accumulating Cd. However, E2 redirected the metal accumulation from the bone and liver to the gill, gut and muscle upon exposure to E2. Cd did not induce the hepatic MT mRNA levels in animals treated with E2. The VTG mRNA levels were also reduced following co-injection of E2 and Cd. However, the kidney responded to Cd exposure by upregulating MT mRNA even in the presence of E2 treatment. In the liver the reduced MT mRNA induction led to a redistribution of CD from MT to non-MT proteins. The toxicological significance of these alterations in Cd handling remains to be determined.