Causal Criteria in Medical and Biological Disciplines: History, Essence, and Radiation Aspects. Report 4, Part 1: The Post-Hill Criteria and Ecolgoical Criteria.

Part 1 of Report 4 is focused on the development and modifications of causal criteria after A.B. Hill (1965). Criteria from B. MacMahon et al. (1970-1996), regarded as the first textbook for modern epidemiology, were considered, and it was found that the named researchers did not offer anything new despite the frequent mention of this source in relation to the theme. A similar situation emerged with the criteria of M. Susser: the three obligatory points of this author, "Association" (or "Probability" of causality), "Time order," and "Direction of effect," are trivial, and two more special criteria, which are the development of "Popperian Epidemiology," i.e., "Surviability" of the hypothesis when it is tested by different methods (included in the refinement in Hill's criterion "Consistency of association") and "Predictive performance" of the hypothesis are more theoretical and hardly applicable for the practice of epidemiology and public health. The same restrictions apply to the similar "Popperian" criteria of D.L. Weed, "Predictability" and "Testability" of the causal hypothesis. Although the universal postulates of A.S. Evans for infectious and noninfectious pathologies can be considered exhaustive, they are not used either in epidemiology or in any other discipline practice, except for the field of infectious pathologies, which is probably explained by the complication of the ten-point complex. The little-known criteria of P. Cole (1997) for medical and forensic practice are the most important. The three parts of Hill's criterion-based approaches are important in that they go from a single epidemiological study through a cycle of studies (coupled with the integration of data from other biomedical disciplines) to re-base Hill's criteria for assessing the individual causality of an effect. These constructs complement the earlier guidance from R.E. Gots (1986) on establishing probabilistic personal causation. The collection of causal criteria and the guidelines for environmental disciplines (ecology of biota, human ecoepidemiology, and human ecotoxicology) were considered. The total dominance of inductive causal criteria, both initial and in modifications and with additions, was revealed for an apparently complete base of sources (1979-2020). Adaptations of all known causal schemes based on guidelines have been found, from Henle-Koch postulates to Hill and Susser, including in the international programs and practice of the U.S. Environmental Protection Agency. The Hill Criteria are used by the WHO and other organizations on chemical safety (IPCS) to assess causality in animal experiments for subsequent extrapolation to humans. Data on the assessment of the causality of effects in ecology, ecoepidemiology, and ecotoxicology, together with the use of Hill's criteria for animal experiments, are of significant relevance not only for radiation ecology, but also for radiobiology.

[Dose-Response Dependences for Frequency of RET/PTC Gene Rearrangements in Papillary Thyroid Carcinoma after Irradiation. Simple Pooling Analysis of Molecular Epidemiological Data].

On the basis of all possible publications on the theme included in the previously formed base of sources on molecular epidemiology of RET/PTC rearrangements in thyroid papillary carcinoma a pooled analysis ("simple pooling data") on determination of the dose-effect dependences for RET/PTC frequency in radiogenic carcinomas of various irradiated groups was performed. (They are groups subjected to radiotherapeutic exposure, residents near the Chernobyl nuclear power plant (CNPP) and victims of nuclear bombing). The tendency to Pearson linear correlation (r = 0.746; p = 0.148) between the frequency of RET/PTC and the estimated dose on thyroid in the regions affected by the CNPP accident was revealed. But this tendency was recognized to be random owing to abnormally low values of the indicator for the most contaminated Gomel region. The method tentatively called "case-control" showed reliable differences in thyroid dose values for carcinomas with RET/PTC and without those. The versatility of changes was found: the lack of RET/PTC for radiotherapeutic impacts was associated with higher doses, whereas in case of the CNPP accident and for nuclear bombing victims it was the opposite. Probably, in the first case the "cellular cleaning" phenomenon after exposure to very high doses took place. Search of direct Pearson correlations between average/median thyroid doses on groups and RET/PTC frequency in carcinomas of these groups showed a high reliability for the dose-effect dependences- at the continuous dose scale (for RET/PTC in total and RET/PTC1 respectively: r = 0.830; p = 0.002 and r = 0.906; p = 0.0003); while there was no significant correlation received for RET/PTC3. When using the weighting least square regression analysis (proceeding from the number of carcinomas in samples), the specified regularities remained. Attempts to influence the strength of correlation by exception ofthe data of all the samples connected with the accident on the CNPP did not significantly reduce the strength of associations for RET/PTC in total. On the basis of ordinal scale doses (background, "low" (0.1 Gy), "middle" (0.1-1 Gy) and "large" (1-10 Gy) dose) also found was a significant correlation (Spearman) with the dose for the frequency RET/PTC in total (r = 0.736; p = 0.0098), but for certain types of rearrangements the results were reverse to the previous analysis (the effect was significant only for the RET/PTC3: r = 0.731; p = 0.024). The linear dose-response trends of the Cochrane-Armitage-test for the frequency of RET/PTC in total, RET/PTC1 and RET/PTC3 depending on the dose to the thyroid in the ordinal scale were registered (p, respectively: < 0.0001 < 0.0001 and 0.007). Thus; after more than 20 years of the molecular and epidemiological research of RET/PTC in thyroid radiogenic carcinomas the comprehensive evidence of the dose-effect dependence existence indicating a real relationship between the studied parameters and a radiation factor was obtained for the first time.

[Pooled Analysis of RET/PTC Gene Rearrangement Rate in Sporadic and Radiogenic Thyroid Papillary Carcinoma].

The database of publications on molecular epidemiology of RET/PTC rearrangements in sporadic and radiogenic thyroid papillary carcinoma has been formed (197 sources at the end of 2014; coverage of 100%). Based on this database a pooled analysis of data on the rates of RET/PTC1, RET/PTC3 and RET/PTC in total was conducted. Statistical approach involves a simple pooling, as well as calculations on the models of random and fixed effects. Since almost all the strata were characterized by heterogeneity, simple pooling and random effect models were adequate. Calculations using both models led to almost identical results. For rates of RET/PTC1, RET/PTC3 and RET/PTC in total with respect to formed carcinoma striations the following values (pooling, in %) were obtained: sporadic, total--13.2; 8.9; 21.2; sporadic, adults--13.3; 9.9; 21.1; sporadic, children--22.4; 17.5; 44.5; radiogenic, total--20.9; 20.3; 40.4; radiotherapy (exposure in childhood)--31.1; 11.8; 42.5; children affected after the Chernobyl accident--19.9; 23.6; 46.1; radiological incidents (exposure in adulthood)--19.9; 7.7; 18.4. Statistically proven is the reliability of differences of carcinoma indicators for children compared with adults (both sporadic and radiogenic tumors) and for radiogenic cancer compared with sporadic. The greatest increase in rate after irradiation was found for RET/PTC1, previously characterized in vitro as one of radiogenic types of RET/PTC.

[RET/PTC Gene Rearrangements in the Sporadic and Radiogenic Thyroid Tumors: Molecular Genetics, Radiobiology and Molecular Epidemiology].

A review of molecular genetic, radiobiological and molecular epidemiological studies of gene (chromosome) rearrangements RET/PTC in the cells of the thyroid gland as well as the laws in relation to radiation exposure in vitro, in vivo and human populations identified with them are submitted. The data on the c-RET gene and its chimeric constructs with the gene-donors (RET/PTC rearrangements) are considered. The information about the history of the RET/PTC discovery, their types, carcinogenic potential and specificity both to tumor and non-tumor thyroid disease especially for papillary thyroid carcinoma are provided. The data (seven studies) on the induction of RET/PTC after irradiation of tumor and normal thyroid cells in vitro and mice are reviewed. The mechanisms of RET/PTC induction may be associated with DNA double strand breaks and oxidative stress. Some information (three publications) about the possibility of RET/PTC induction by low doses of radiation with low LET (to 0.1 Gy) is given and it is concluded that their potential evidentiary is generally weak. The achievements in the molecular epidemiology of RET/PTC frequency for exposed and unexposed cohorts are stated. At the same time it is noted that, despite the vast array. of data accumulated from 30 countries of the world and more than 20 years of research, the formed provisions are weakly confirmed statistically and have no base corresponding to the canons of evidence-based medicine. The possibility of use of the RET/PTC presence or their frequencies as markers of the papillary thyroid carcinomas and, specifically, their radiogenic forms, is considered. In the first case the answer may be positive, while in the second, the situation is characterized by uncertainty. Based to the above mentioned we came to a conclusion about the need of a pooled or meta-analysis of the totality of the published data.

[Search problems of human radiation protection in the world of genetics of aging].

Currently, the urgency for protection from negative effects of radiation in the range of low and medium dose where classic radioprotectors are ineffective is increased. In this respect it seems promising to study the molecular pathways that increase, on the one hand, the stability of the genome against radiation damage (inducers of carcinogenesis), and, on the other hand, elevate the radiation sensitivity of cell populations in order to eliminate potentially carcinogenic cells. This approach requires modification of cascade mechanisms of signal transduction to apoptosis and responses to DNA damage. Research plan is similar to the Genetics of Aging, where a number of hypotheses about the mechanism of aging have been proposed, including a decrease in the stability of the genome to external influences. Proceedings of the 2nd International Conference "The genetics of aging and longevity" (Moscow, April 2012) demonstrated, however, that patterns of aging mechanisms identified in model animals (nematodes, drosophila and mice) are far from the possibility of their practical application. Discovered genes that may be responsible for life expectancy (stress-inducible protein and other components of the signal transduction cascade, as well as suppressors and inducers) rarely find significance in the study of the genomes of centenarian cohorts. This may be due to the difficulty in transferring molecular genetic patterns from model objects to large mammals, including humans, with respect to systems of signal transduction. This point must be taken into account during the search for a new generation of radioprotective agents that promote anti-carcinogenic potential of human cells exposed to radiation at low and moderate doses. It may be necessary to search for such tools in large laboratory animals and in human tissue cultures obtained through genetic engineering or cloning.

[Antiradiation effects of lactoferrin].

The propose of these examination was studied antiradiation effect of lactoferrin (LF). It was determined, that subcutaneous injection LF (65 or 300 mkg/kg) guinea-pigs irradiated after 1-14 days after of total gamma-irradiation (2.5 Gr) had therapeutically effect attached to the bone marrow form of acute radiation disease (ARD). This effect was explained in increase of survive the animals with 53.8% to 92.8% and stimulation erythropoiesis, judge by number of reticulocytes and increase neutrophiles (12 days ARD) and lymphocytes (12 and 16 days ARD) by comparison with control animals. LF had no effect attached to intestinal form of acute radiation disease by mice. The injection of LF before total irradiation rats of electrons (energy 25 MeV) in doses 255 Gr decreased expression of some symptom of cerebral form of acute radiation disease: prolong time of beginning convulsive period with 5.7 hours to 14.1 hours and duration of life with 7.7 hours to 15.3 hours. LF had antioxidative activity.

[Different trend for changing of mammalian plasma (serum) antioxidant activity after irradiation at large and low-doses].

One of the path for research of antioxidant status in vivo is the determination of blood plasma (serum) antioxidant activity (AOA). AOA is an integral parameter for the contribution of different antiradical defense components which particular nomenclature is determined by a used method of definition. In the present work AOA is studied with usage of a system of suppression in vitro by blood plasma (serum) of a Fe(2+)-induced oxidation of a substrate from egg yolk lipoproteins. The contribution of separate components of a blood plasma to an integral index of AOA for human blood serum is studied. Urine acid, albumin, reduced glutathione, ascorbat, heparin, peroxidase and catalase had no activity. Apo-beta-lipoprotein, vitamin E, ceruloplasmin and transferring are introduce, possibly, the main contribution to AOA. The significant decrease of AOA was revealed after acute irradiation by gamma-rays of dogs (4.05 Gy) and mice (6 Gy). The irradiation of mice at low-dose (0.2 Gy), vice-versa, result to stimulation of AOA with clear peak value on 5 day. The possible reasons of quality difference for effects of large and low dose radiation with reference to antioxidant system of organism are discussed.

[The absence of the facts connected with the genomic instability after the irradiation in low doses by radiation with low LET].

In the review which is a brief account of more complete document (Koterov A.N. // Int. J. Low Radiat. 2005. V. 1. No. 4. P. 376-451) the data of world researches devoted to a phenomenon of radiation-induced genomic instability (RIGI) are considered. The purpose of the review is the definition of the bottom limit of radiation doses which induced of RIGI in experiments at different methodical approaches (irradiation in vitro, in vivo, in utero, bystander effect and transgeneration effects of radiation). The action only radiation with low LET is examined. Among several hundreds works wasn't revealed any fact, when RIGI induced by low doses irradiation (up to 0.2 Gy) for normal cells and for organism left from maternal womb. Six exceptions are revealed which are named as "apparent" so in all cases the abnormal, unstable, defective objects or ambiguous final parameter were used. Thus, RIGI at low doses of radiation with low LET is a myth.

Antioxidant spectrum of blood serum and its peculiarities in children.

We compared the contribution of high- and low-molecular-weight antioxidants into total antioxidant activity of blood serum in children and adults. Ten serum samples from children aged from 3 months to 12 years and 6 serum samples from adults were fractionated by chromatography and antioxidant activity and the contents of transferrin and ceruloplasmin were measured in total serum and individual chromatographic fractions. It was found that total antioxidant activity of the serum from children measured in the system of yolk lipoproteins considerably surpassed that in adults. Moreover, in adults the major part in serum antioxidant activity is played by a fist identified high-molecular-weight fraction (600 kDa) and a 67 kDa fraction containing ceruloplasmin and transferrin. Serum antioxidant activity in children was determined only by the high-molecular-weight peak not containing ceruloplasmin and transferrin, which was probably due to significantly lower serum transferrin content in children compared to adults.

[Bcl-2 apoptosis regulating proteins in mouse tissues after exposure to 20 cGy of gamma-radiation]. / Belki-reguliatory apoptoza is semeistva bcl2 v tkaniakh myshei posle vozdeistviia gamma-izlucheniia.v doze 20 sGr.

A pronounced increase of Bcl-2 content was observed in thymus of mice exposed to 20 cGy of gamma-radiation 1 and 24 hours after the exposure; six days after the exposure the bcl-2 content lowered to the control one. No changes in bcl-2 level were revealed in mice liver during the period of observation. A level of Bax protein was stable in mice thymus during 60 days of observation.

[The radiation-modifying capacity of xenogenic apotransferrin for the number of endogenous colony forming units in the spleen of irradiated mice]. / Radiomodifitsiruiushchie svoistva ksenogennogo apotransferrina po pokazateliu chisla éndogennykh KOE v selezenke obluchennykh myshei.

After intraperitoneal injection of 100 or 198 mg/kg of human serum apotransferrin (apo TF) to mice 1 day before acute exposure to 6 Gy of gamma-radiation, the number of endogenous CFU in spleen (CFUs) increased 2.5 or 2.6 times respectively. At a dose fo 10 mg/kg of the protein only an increasing tendency was found, whereas a dose of 1 mg/kg was inefficient. A dose of 100 mg/kg of BSA did not show any effect suggesting that non-specific immune response to alien antigen did not contribute to apo TF radiomodifying action. The following mechanisms of the apoTF radiomodifying effect are discussed: 1) the ability of the protein to inactivate Fe3+ ions that reduces the consequences of radiation oxidative stress; 2) the stimulation of proliferation of the exposed bone marrow cells by activation of Fe3+ transport or by Ca2+ mediated mechanism of mitogen signal transduction; 3) changing in the content and ratio of cyclic nucleotides by apo TF stimulation of Ca-calmodulin-dependent phosphodiesterase.

[Radioadaptive response in vitro of unstimulated rat lymphocytes according to metallothionein test]. / Radioadaptivnyi otvet in vitro nestimulirovannykh limfotsitov krys po metallotioneinovomu testu.

It was shown that 24 h after acute action of gamma-radiation in vitro on unstimulated rat lymphocytes the metallothionein (MT) level did not change for doses of 0.01; 0.5 and 1.0 Gy, while dosed of 1.75 and 2.5 Gy increased this parameter 2 and 2.6 times on the average. After exposure to 0.01 Gy of adaptive radiation 2 h before exposure to 1.75 Gy of the damaging dose reliably lower (1.5 times on the average) increase in MT content was found. The heterogeneity in display of the radioadaptive response by MT test for various lymphocyte preparations was found. Two possible mechanisms of the obtained changes were discussed. One of them is connected with the selection irradiation death of cells with the initially low MT level, and the other is caused by postradiation MT synthesis as a result of accumulation of the DNA damages and MT genes amplification.

[Parameters of proliferative activity of hematopoietic cells in mice protected from irradiation by indralin in combination with Zn-metallothionein]. / Pokazateli proliferativnoi aktivnosti kletok krovetvornykh organov u myshei, zashchishchennykh ot izlucheniia indralinom v sochetanii s Zn-metallotioneinom.

One day after the irradiation (dose 6 Gy) of mice protected by the injection of Zn-metallothionein (Zn-MT) in doze 8.6 mg/kg, 10-20 min before irradiation, then alpha-adrenomimetic indraline (150 mg/kg) 5-10 min before irradiation the increase in nucleic cell number, [3H] thymidine incorporation, and antioxidant activity in bone marrow in comparison with the control and indraline per se was revealed. In mice protected according to the scheme: Zn-MT in the same doze, then indraline (100 mg/kg) one day after, and then in 5-10 min exposure to 6 Gy it was found more than 9 times increase of endogeneous CFC in spleen on 8th day while indraline per se raised CFC number only 4.8 times. It was found that Zn-MT reduce the indraline acute toxicity. The data on radioprotective activity of monomeric and polymeric Zn-MT forms are submitted.

[Molecular and cellular mechanisms of the adaptive response in eukaryotes]. / Molekuliarnye i kletochnye mekhanizmy adaptivnogo otveta u éukariot.

The mechanisms of adaptive response (AR) which consisted of the increasing of cell and organism resistance to large doses of genotoxic and toxic agents formed after the preliminary influencing of its agents in small doses were discussed. The examples of factors leading to AR (irradiation, radiomimetics, alkylators, H2O2, and hyperthermy) as well as the conditions of its manifestation (in vivo and in vitro, acute and chronic adapting influences) were produced. The AR mechanisms bases on the molecular and cellular levels are the stimulation of protecting system (the synthesis of protecting proteins and antioxidant system activation), the intensification of DNA repair, effect on neurohumoral signal messengers and changes of proliferative activity. On organism level the nonspecific stress-reaction components were added (AKTG and steroid hormones synthesis). It was concluded that no there was no single mechanism of AR to all type of damaging agents. Evidently of that causes of its phenomenon have the complex character. The relatively narrow limits of AR (variability for the cells of different individuals, slight AR in children's cells and full absence of embryo cells) require careful use an AR conformities as the indicator for the professional and referring works.

[Dynamics of malonic aldehyde, transferrin levels and blood antioxidant activity in women with normal pregnancy and pregnancy complicated by toxicosis: effect of eiconol]. / Dinamika urovnia malonovogo dial'degida, transferrina i antioksidantnoi aktivnosti syvorotki krovi u zhenshchin s normal'noi beremennost'iu i beremennost'iu, oslozhnennoi toksikozom: vliianie éikonola.

Malonic dyaldehyde (MDA) and transferrin (TF) content and total antioxidant activity (AOA) were measured in blood of pregnant women with normal pregnancy (27 women) and preeclamptic pregnancy (46 women) at different pregnancy stages (17-28, 29-34 and 35-40 weeks of pregnancy). It was shown that in normal pregnancy AOA increased from 24 weeks of pregnancy with a maximum at 30 weeks, while MDA level did not change significantly through all time of pregnancy. On the contrary, in preeclamptic pregnancy blood AOA did not change significantly, while MDA content increased at the 26-34 weeks of pregnancy with a maximum at 30-34 weeks of pregnancy. Blood TF content in normal pregnancy increased during pregnancy development with a maximum at 30-34 weeks, while in preeclamptic pregnancy TF changed only a little. These data show the important role of TF in the AOA blood status formation in normal pregnancy, and great pathogenetic role of the absence of such an increase of TF at preeclamptic pregnancy in AOA impairment at this condition. Eiconol (omega-3 polyunsaturated fatty acid concentrate from north fish oil) intake by preeclamptic pregnant women drastically decreased blood MDA level (1,5-2 times compared to the women that did not receive eiconol), especially at 29-34 week of gestation. Blood AOA, that was decreased at preeclampsia increased under influence of eiconol intake at all pregnancy stages studied. Eiconol intake greatly increased also the TF blood content that was decreased at preeclampsia. It is supposed that eiconol ability to increase AOA may be explained by its capacity to increase the level of TF--one of the most important compounds of serum blood antioxidant system.

[Adaptation to irradiation in vivo]. / Adaptatsiia k oblucheniiu in vivo.

The data about the increasing of radioresistance of cells and organism after the acute, chronic and prolonged irradiation in vivo were presented. The possible mechanisms of adaptation to irradiation connected with selection, stimulation of proliferative activity, forming of protecting system (protected proteins, antioxidant enzymes e.a.), activation of DNA repair and cAMP-, and Ca(2+)-dependent phosphorylation systems were observed. The conclusion about complex mechanism of radiation adaptation was drawn.

[The effect of zinc metallothionein on lipid peroxidation in rodent bone marrow cells]. / Vliianie tsink-metallotioneina na perekisnoe okislenie lipidov v kletkkah kostnogo mozga gryzunov.

It was shown, that rat hepatic Zn-metallothionein (Zn-MT) depressed in vitro Fe3+/ADP-induced lipid peroxidation (LP) in rat bone marrow cells and increased its viability after pro-oxidant's influences. ZnCl2 and its combination with cysteine also decreased LP products level, but less and did not alter viability. On 9-th day after gamma-irradiation (6 Gy) in mice protected with Zn-MT the LP products level in bone marrow was decreased in considerable extent.

[The possible mechanism of the antiradiation effect of metallothionein: the stimulation of DNA replicative synthesis and of bone marrow cell proliferation]. / Vozmozhnyi mekhanizm protivoluchevogo éffekta metallotioneina: stimuliatsiia replikativnogo sinteza DNK i proliferatsii kletok kostnogo mozga.

Zn-MT and less apo MT (4-100 mkg/ml) in vitro stimulated the [3H]-thymidine incorporation in mice bone marrow cells, but ZnCl2, its combination with cysteine and mixture (ZnCl2 + cystine + albumin) inhibited DNA synthesis. In protected with Zn-MT (1-4 mg/kg) 10-15 min before irradiation (6 Gy) mice on 9th day after influencing the increasing of DNA replicative synthesis level and nuclear cell number in bone marrow were observed.

[The diverse effects of the combined action of cadmium chloride and ionizing radiation on the content of metallothioneins in mouse bone marrow and liver]. / Razlichnye éffekty sochetannogo deistviia khloristogo kadmiia i ioniziruiushchei radiatsii na soderzhanie metallotioneinov v kostnom mozge i pecheni myshei.

It was shown that introduction of cadmium chloride (0.75 mg/kg Cd2+) in combination with gamma irradiation (8.5 Gy) of mice increases the level of metallothioneines (MTs) in the bone marrow and liver of mice. The maximum effect was observed in 24-30 h after the performance. Similarly, irradiation with the doses of 3 to 10 Gy resulted in an increase in the contents of both MT isoforms (MT1 and MT2) in the bone marrow in 24 h. A combined action of gamma irradiation and a heavy metal caused an additive effect on the MT content in the bone marrow, whereas the MT content in liver was 2 times lower than that predicted theoretically. A possible mechanism of the discovered phenomenon was discussed. Supposedly, it is associated with different degrees of the radiation-induced inhibition of the MT1 and MT2 expression by cadmium ions.