To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii.

Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the "sizer" in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.

Morphological changes in melanized and non-melanized Cryptococcus neoformans cells post exposure to sparsely and densely ionizing radiation demonstrate protective effect of melanin.

There is a need for novel and effective prophylactic treatments and radioprotective materials to protect civilians and military personnel from ionizing radiation in contaminated environments. Melanin, a naturally occurring, ubiquitous pigment, has been shown to confer radioresistance, acting as a potential radioprotective agent. We have demonstrated that melanized Cryptococcus neoformans (CN) cells had improved survival post ionizing irradiation than non-melanized ones. The goal of this study was to identify morphological changes in melanized and non-melanized CN cells following irradiation with densely-ionizing deuterons and alpha particles relative to sparsely-ionizing gamma radiation. We observed significant differences between the melanized and non-melanized CN cellular ultrastructure following irradiation. Melanized CN cells were relatively resistant to mid and max-dose levels of alpha particles and deuterons irradiation. Following irradiation the capsule was stripped, but the cell wall was intact and structural integrity was maintained. At the maximum dose, cytoplasmic vacuolization, and mitochondrial swelling started to occur. In contrast, the non-melanized CN strain was sensitive to the mid-dose radiation. Non-melanized cells presented two morphologies: small condensed, and swollen, lacking structural integrity. This morphological investigation provides the first direct evidence of the radioprotective properties of melanin in CN cells subjected to high RBE and high LET ionizing radiation.

Stable isotope compounds - production, detection, and application.

Stable isotopes are used in wide fields of application from natural tracers in biology, geology and archeology through studies of metabolic fluxes to their application as tracers in quantitative proteomics and structural biology. We review the use of stable isotopes of biogenic elements (H, C, N, O, S, Mg, Se) with the emphasis on hydrogen and its heavy isotope deuterium. We will discuss the limitations of enriching various compounds in stable isotopes when produced in living organisms. Finally, we overview methods for measuring stable isotopes, focusing on methods for detection in single cells in situ and their exploitation in modern biotechnologies.

Large deuterium isotope effects and their use: a historical review.

Isotope effects are differences in the properties of the isotopes of an element resulting in different reaction rates of a corresponding compound, in equilibrium constants and in the spectra. Shortly after the discovery of stable isotopes of hydrogen, oxygen, and carbon, Jacob Bigeleisen formulated a theory of isotope effects and calculated possible maximum values. Large isotope effects of (2)H (deuterium) against (1)H (protium) were seen to possibly influence interpretations of reaction mechanisms if corresponding labelling is used. Much work was invested to ensure the safety of deuterium use in men in spite of the large isotope effect. On the other hand, large deuterium isotope effects gave rise to several practical applications. Examples are the enhancement of the stability of some technical products against oxidative and against hydrolytic degradation (oils, pharmaceuticals) as well as alterations of the detoxification metabolism of pharmaceuticals in vivo.

Pharmaco-thermodynamics of deuterium-induced oedema in living rat brain via 1H2O MRI: implications for boron neutron capture therapy of malignant brain tumours.

In addition to its common usage as a tracer in metabolic and physiological studies, deuterium possesses anti-tumoural activity and confers protection against gamma-irradiation. A more recent interest in deuterium emanates from the search for alternatives capable of improving neutron penetrance whilst reducing healthy tissue radiation dose deposition in boron neutron capture therapy of malignant brain tumours. Despite this potential clinical application, deuterium induces brain oedema, which is detrimental to neutron capture therapy. In this study, five adult male rats were titrated with deuterated drinking water while brain oedema was monitored via water proton magnetic resonance imaging. This report concludes that deuterium, as well as deuterium-induced brain oedema, possesses a uniform brain bio-distribution. At a steady-state blood fluid deuteration value of 16%, when the deuterium isotope fraction in drinking water was 25%, a mean oedematous volume change of 9 +/- 2% (p-value <0.001) was observed in the rat brain-this may account for neurological and behavioural abnormalities found in mammals drinking highly deuterated water. In addition to characterizing the pharmaco-thermodynamics of deuterium-induced oedema, this report also estimates the impact of oedema on thermal neutron enhancement and effective dose reduction factors using simple linear transport calculations. While body fluid deuteration enhances thermal neutron flux penetrance and reduces dose deposition, oedema has the opposite effect because it increases the volume of interest, e.g., the brain volume. Thermal neutron enhancement and effective dose reduction factors could be reduced by as much as approximately 10% in the presence of a 9% water volume increase (oedema).

Deuterium isotope effect on the metabolism of the flame retardant tris(2,3-dibromopropyl) phosphate in the isolated perfused rat liver.

The metabolism of tris(2,3-dibromopropyl) phosphate (Tris-BP) was compared with that of completely deuterated Tris-BP (D15-Tris-BP) in an isolated, recirculating rat liver perfusion system in order to determine the relative quantitative importance of two different biotransformation pathways of Tris-BP: (i) cytochrome P450-mediated metabolism and (ii) GSH S-transferase-mediated metabolism. To accomplish this we quantitated the biliary excretion of S-(3-hydroxypropyl)glutathione (GSOH) as a marker metabolite for cytochrome P450-mediated metabolism and that of S-(2,3-dihydroxypropyl) glutathione (GSOHOH) as a marker metabolite for GSH S-transferase-mediated metabolism. Complete deuterium substitution of Tris-BP significantly decreased the formation of GSOH, whereas there was no effect on the formation of GSOHOH. Because our previous studies showed a large decrease in genotoxicity of D15-Tris-BP compared to Tris-BP, the present results support our hypothesis that cytochrome P450-mediated metabolism is responsible for the genotoxic effects of Tris-BP in the rat liver.

[Comparison of the teratogenic properties manifested in BALB c mice, by diphenylhydantoin and deuterated diphenylhydantoin]. / Comparaison des propriétés tératogènes manifestées, chez les souris de race BALB-c, par la diphénylhydantoïne et la diphénylhydantoïne deutérée.

The aim of this study was to investigate the role of an arene oxide pathway in the teratogenicity displayed by DPH, a highly effective antiepileptic agent. This approach was carried out by comparing the teratogene potential of DPH and of p2-H-DPH administrated at days 8, 9 and 12 of gestation. The present findings support the hypothesis that substitution of protium by deuterium at the para position of one of the phenyl rings, which favours an arene oxide pathway, causes an increase in some to the teratogenic effects of DPH.

Heavy water (D2O)-induced shape changes, movements and F-actin redistribution in human neutrophil granulocytes.

Heavy water (D2O) induces characteristic shape changes and a distinct type of movement in human neutrophil granulocytes. In contrast to front-tail polarity as evoked by chemotactic peptides and microtubule-disassembling agents, D2O-based media produce non-polar neutrophils with many small or long surface projections. This phenotype is similar to that elicited by both phorbol myristate acetate and diacylglycerols, but the surface projections are smaller and more densely placed and are often associated with a single large projection. D2O-induced non-polar cells with surface projections perform continuous shape changes without front-tail polarity and without the unidirectional movement and cytoplasmic streaming seen in cells with front-tail polarity. Some of the cells show circus movements of a large projection indicating circular polarity. In neutrophils suspended in D2O, F-actin is shifted to the cell periphery, mainly into the surface projections of activated cells. The D2O-induced effects are reversed in H2O-based medium. D2O is dominant over the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), colchicine and taxol in that the combined action of D2O with any of these agents results in the D2O-induced phenotype. In contrast, cytochalasin B alone and in combination with fMLP induces a considerable decrease of non-polar cells and an increase of spherical cells similar to non-stimulated cells in H2O-based medium. Earlier studies indicated that D2O acts on microtubules. Our results suggest that D2O may act on the microfilament system. Neutrophils suspended in D2O-based medium may represent a useful model to study the relationship between shapes, movements, and particular functions of these cells.

Comparative neurotoxicity and pyrrole-forming potential of 2,5-hexanedione and perdeuterio-2,5-hexanedione in the rat.

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, reacts with protein amines to form alkylpyrrole adducts. Pyrrolylation of neurofilament protein may be the initiating molecular event in 2,5-HD neuropathy. The present study compares the neurotoxic and pyrrole-forming potentials of 2,5-HD with those of perdeuterio-2,5-HD ([D10]-2,5-HD) in the rat. Due to a requirement for C-H bond breaking in the reaction mechanism, the latter derivative was expected to exhibit a primary isotope effect, thus forming the pyrrole at a slower rate. In vitro studies confirmed that [D10]-2,5-HD pyrrolylated protein at only one-third of the initial rate seen with native 2,5-HD. Prolonged incubation resulted in similar pyrrole concentrations with both derivatives. Adult, male Wistar rats were administered daily (5 days/week) ip doses of either 3.5 mmol 2,5-HD or [D10]-2,5-HD/kg/day for 17 days or 2.5 mmol/kg/day for 38 days. At termination, animals administered 2,5-HD and [D10]-2,5-HD exhibited 27 and 8% body weight loss, respectively. Moderate to severe hindlimb paralysis was present in the 2,5-HD groups while only mild effects were seen in [D10]-2,5-HD-dosed rats. Neuropathological changes were prominent in spinal cord sections from 2,5-HD-treated animals, while no effects were present in rats given the deuterated derivative. Pyrrole adduct concentrations in serum and axonal cytoskeletal proteins from 2,5-HD-treated animals were two- to threefold higher than in rats given equimolar doses of [D10]-2,5-HD. Levels of covalent crosslinking of axonal cytoskeletal proteins (assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) appeared to correlate with pyrrole concentrations. Tissue concentrations of each diketone isomer were not significantly different, indicating similar uptake of native and deuterated 2,5-HD. Mass spectrometry revealed rapid back exchange of the terminal (methyl) but not of the internal (methylene) deuteriums of [D10]-2,5-HD in vivo. These findings support an absolute requirement for pyrrole formation in gamma-diketone neurotoxicity.

Use of stable isotopes to determine compliance.

The natural variation in the abundance of stable isotopes of light elements (C, H, O, N) in humans is less than 0.5%. Much larger variations can be induced through administration of drugs labeled with enriched isotopes of these elements. Such labels thus can be used as tracers of compliance. Variations of several percent can be generated without adverse physiological effect. An example is presented of the use of deuterium oxide as a tracer, and a scheme of sampling moisture in expired air is proposed, in which the subject can easily collect a sample and present it for rapid analysis. Tracer levels can be used to estimate the degree of compliance. The labeled compound can be selected to match the drug being tested with respect to residence time in the body. Except for deuterium, the cost of such stable isotope tracers is at present prohibitive. The main problem with the use of stable isotopes in compliance studies is the cost of the tracer, a biologically acceptable substance labeled with a rare-stable isotope. Our original experiments were carried out using 13C-glucose, which costs at present about $600/g. To carry out a tracer experiment on an individual, approximately 10 mg of uniformly labeled tracer would be administered, which would result in a 5% shift of the 13C/12C ratio, which is easily detectable. To minimize the cost of such a study, we proposed utilizing the cheapest enriched light isotope that is available, namely, deuterium. Using a ratio-detecting mass spectrometer in which a sample is compared with a standard, it is possible to detect enrichment or depletion of deuterium with respect to 1H at the level of 0.1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological hazards of deuterium oxide as a cardioplegic agent.

To determine the cardio-protective effect of heavy water on the ischemic myocardium, a thoracotomy was performed on 18 mongrel dogs. The animals were connected to the extracorporeal circulation in a standardized experimental procedure. Following total cardiopulmonary bypass, 2,000 ml of a standard cardioplegic solution (LK 352) was infused at the aortic root of 10 dogs, which served as controls (group I), and the same solution containing 20% of 99.8% deuterium oxide was given at the aortic root of the remaining animals (group II). At the end of 60 minutes of ischemia, 1,000 ml of the solutions was again administered at the aortic root of the corresponding animals. Myocardial biopsies were taken from the apex of the left ventricle of each dog before cardiopulmonary bypass, immediately after the infusion of the cardioplegic solutions, following 90 minutes of ischemia, and after 30 minutes of reperfusion, and studied ultrastructurally. Whereas the ultrastructure of the myocardium of group I was well preserved at the end of the ischemic period, deuterium-oxide-treated hearts showed extensive focal and global myofilamentolysis and lysis of whole myocytes. Structural damage to glycogen, nuclear chromatin dispersal, severe intracellular edema and complete rupture of the intercalated discs were characteristic findings. At the end of ischemia, all the hearts of group I could be resuscitated. During the ischemia, all the hearts of group II developed into stone hearts. Biochemical studies on a second series showed a higher ATP depletion and a significantly higher lactate accumulation in group II than in group I.(ABSTRACT TRUNCATED AT 250 WORDS)

Abolishment of the Ranvier node excitability under quasi-total deuteration and rhythmic stimulation.

The isolated nerve fibre, when quasi-totally deuterated, loses its excitability after about 15' of rhythmic stimulation at frequencies of 150-200 Hz. It is only on internal rehydration that excitability is recovered at high frequencies of stimulation. An excitation-energy coupling is suggested to exist, which appears to be more obvious at high frequencies of stimulation and strongly affected by the proton-deuteron exchange in the intracellular medium.

Studies of a deuterium isotope effect in carcinogenesis by N-nitroso-N-alkylurethanes in rats.

Nitroso-N-methylurethane and nitroso-N-ethylurethane were administered to groups of 20 female F344 rats in corn oil solution by gavage. Each rat received once a week 0.2 ml of solution containing 7 mg/ml or 1.75 mg/ml of the methyl compound or 8 mg/ml or 2 mg/ml of the ethyl compound for 20 weeks. In parallel with these treatments additional groups of rats were given equimolar dose of nitrosomethylurethane and nitrosoethylurethane fully labeled with deuterium in the N-alkyl groups. All animals were allowed to die naturally. The total doses received by the rats were 0.2 mmol at the higher concentration and 0.05 mmol at the lower concentration. Almost all of the treated rats died with papillomas and carcinomas of the forestomach (non-glandular stomach), and the other induced tumors of significance were carcinomas and papillomas of the esophagus in rats given the 7 mg/ml dose of nitrosomethylurethane, both labeled and unlabeled. There was no significant difference in tumor incidence or rate of mortality from tumors that would indicate a difference in carcinogenic effectiveness between the nitrosoalkylurethanes and their deuterium-labeled counterparts. Apart from the esophageal tumors induced only by nitrosomethylurethane at the higher dose, there was no significant difference in carcinogenic effectiveness between the methyl and ethyl nitrosourethanes.

Nephrotoxicity and hepatotoxicity of chloroform in mice: effect of deuterium substitution.

Chloroform (CHCl3) produces renal and hepatic damage in humans and experimental animals. Deuterium-labeled chloroform (CDCl3) has been reported to be less hepatotoxic than CHCl3 in rats. However, this isotope effect has not been determined in other species or in extrahepatic tissues. In this investigation, the effect of deuterium substitution on the nephrotoxicity and hepatotoxicity of CHCl3 was quantified in male ICR mice. Renal and hepatic damage were determined 24 h after administration on various doses of CHCl3 or CDCl3. Liver damage was estimated by measuring serum glutamic-pyruvic transaminase (SGPT) activity. Nephrotoxicity was evaluated by measuring blood urea nitrogen (BUN) and in vitro renal cortical accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA). Dose-related hepatotoxicity and nephrotoxicity were observed after administration of CHCl3 and CDCl3. CDCl3 produced less liver damage than CHCl3 in mice, suggesting that mouse liver metabolizes CHCl3 by the same mechanism as rat liver. CDCl3 was also less toxic to kidneys than CHCl3, suggesting that the kidney may metabolize CHCl3 in the same manner as the liver

Effects of temperature and deuterium oxide on crustacean stretch receptor.

1. The receptor potential was recorded intracellularly from the isolated stretch receptor neuron of crayfish. Total substitution of D2O for H2O in the bathing solution reversibly decreased the amplitude of the receptor potential to a level of 34% of the control. The input resistance of the neuron was slightly increased by the D2O treatment, while the resting potential and the input capacity were virtually unchanged. The viscoelastic property of the receptor muscle was not altered by the D2O substitution. Thus, D2O seems to inhibit directly the activation process of the receptor potential. 2. Temperature change over the range of 25-5 degrees C did not affect the amplitude of receptor potential. However, the input resistance of the neuron was increased about twofold by the lowering of temperature from 25 to 5 degrees C. 3. We conclude that when the temperature is lowered, the receptor current is diminished, but this decline is just about compensated for by the increase in membrane resistance, resulting in a near constancy of the receptor potential. This is the cellular basis for the relative constancy of the transduction process of the stretch receptor in the face of large temperature changes. On the other hand, in the case of D2O substitution the compensation mechanism of the resistance increase does not work, and the sensory transduction is inhibited substantially.

Influence of deuterium on the spleen immune system.

Following administration of deuterium to white rats changes of dysimmunitary-immunitary depression occurred. At the beginning the changes involved the whole spleen parenchyma, with marked lymphocyte depopulation of the red pulp and a decrease of the lymphod follicles. After about 4 - 7 days important amorphous deposits were formed, located exclusively perifollicularly and only in the spleen. The authors suggest the dysimmunitary globulinic-paraamyloidic nature of these deposits which, in their opinion, are produced by deuterium through a chemical mechanism. The differential diagnosis was only made with Gamma-Gandy nodules, because no such changes are found in pathology. It is not known why this deposition takes place exclusively in the spleen and especially around the lymphoid follicles. The authors raise the question whether some methods could be found which might direct the immunitary blockade to certain organs.