Anatoly N. Varaksin

Some considerations concerning the theory of combined toxicity: a case study of subchronic experimental intoxication with cadmium and lead.

Rats were exposed intraperitoneally (3 times a week up to 20 injections) to either Cadmium and Lead salts in doses equivalent to their 0.05 LD50 separately or combined in the same or halved doses. Toxic effects were assessed by more than 40 functional, biochemical and morphometric indices. We analysed the results obtained aiming at determination of the type of combined toxicity using either common sense considerations based on descriptive statistics or two mathematical models based (a) on ANOVA and (b) on Mathematical Theory of Experimental Design, which correspond, respectively, to the widely recognised paradigms of effect additivity and dose additivity. Nevertheless, these approaches have led us unanimously to the following conclusions: (1) The above paradigms are virtually interchangeable and should be regarded as different methods of modelling the combined toxicity rather than as reflecting fundamentally differing processes. (2) Within both models there exist not merely three traditionally used types of combined toxicity (additivity, subadditivity and superadditivity) but at least 10 variants of it depending on exactly which effect is considered and on its level, as well as on the dose levels and their ratio.

Attenuation of Combined Nickel(II) Oxide and Manganese(II, III) Oxide Nanoparticles’ Adverse Effects with a Complex of Bioprotectors

Stable suspensions of NiO and Mn3O4 nanoparticles (NPs) with a mean (±s.d.) diameter of 16.7 ± 8.2 and 18.4 ± 5.4 nm, respectively, purposefully prepared by laser ablation of 99.99% pure nickel or manganese in de-ionized water, were repeatedly injected intraperitoneally (IP) to rats at a dose of 2.5 mg/kg 3 times a week up to 18 injections, either alone or in combination. A group of rats was injected with this combination with the background oral administration of a “bio-protective complex” (BPC) comprising pectin, vitamins A, C, E, glutamate, glycine, N-acetylcysteine, selenium, iodide and omega-3 PUFA, this composition having been chosen based on mechanistic considerations and previous experience. After the termination of injections, many functional and biochemical indices and histopathological features (with morphometric assessment) of the liver, spleen, kidneys and brain were evaluated for signs of toxicity. The Ni and Mn content of these organs was measured with the help of the atomic emission and electron paramagnetic resonance spectroscopies. We obtained blood leukocytes for performing the RAPD (Random Amplified Polymorphic DNA) test. Although both metallic NPs proved adversely bio-active in many respects considered in this study, Mn3O4-NPs were somewhat more noxious than NiO-NPs as concerns most of the non-specific toxicity manifestations and they induced more marked damage to neurons in the striatum and the hippocampus, which may be considered an experimental correlate of the manganese-induced Parkinsonism. The comparative solubility of the Mn3O4-NPs and NiO-NPs in a biological medium is discussed as one of the factors underlying the difference in their toxicokinetics and toxicities. The BPC has attenuated both the organ-systemic toxicity and the genotoxicity of Mn3O4-NPs in combination with NiO-NPs.

Further development of mathematical description for combined toxicity: A case study of lead–fluoride combination

Highlights • Rats were exposed during 6 weeks to repeated i.p. injections of lead acetate, sodium fluoride or both in doses 0.05 LD50.• The development of intoxication was estimated quantitatively with 54 functional, biochemical and morphometric indices.• For mathematical description of effects a response surface regression model containing linear and cross terms was used.• 10 types of combined action were found thus confirming the result of the similar study on Pb and Cd.• Special attention is given to indices on which combined toxics produce opposite effects.

Further development of the theory and mathematical description of combined toxicity: An approach to classifying types of action of three-factorial combinations (a case study of manganese–chromium–nickel subchronic intoxication)

For characterizing the three-factorial toxicity, we proposed a new health risk-oriented approach, the gist of which is a classification of effects depending on whether a binary combined toxicitys type remains virtually the same or appears to be either more or less adverse when modeled against the background of a third toxic. To explore possibilities of this approach, we used results of an experiment in which rats had been injected ip 3 times a week (up to 20 injections) with a water solution of either one of the toxics (Mn, Ni or Cr-VI salts) in a dose equivalent to 0.05 LD50, or any two of them, or all the three in the same doses, the controls receiving injections of the same volume of distilled water (4mL per rat). Judging by more than 30 indices for the organisms status, all exposures caused subchronic intoxication of mild to moderate strength. For each two-factorial exposure, we found by mathematical modeling based on the isobolograms that the binary combined subchronic toxicity either was of additive type or departed from it (predominantly toward subadditivity) depending on the effect assessed, dose, and effect level. For the three-factorial combination, different classes of effects were observed rather consistently: class A - those regarding which the third toxics addition made the binary toxicity type more unfavorable for the organism, class B - those regarding which the result was opposite, and class C - those regarding which the type of binary combined toxicity on the background of a third toxic virtually remained the same as in its absence. We found a complicated reciprocal influence of combined metals on their retention in kidneys, liver, spleen and brain which might presumably be one of the possible mechanisms of combined toxicity, but the lack of an explicit correspondence between the above influence and the influence on toxicity effects suggests that this mechanism is not always the most important one. The relevance of the proposed classification to health risk analysis and management is briefly discussed.

In vivo toxicity of copper oxide, lead oxide and zinc oxide nanoparticles acting in different combinations and its attenuation with a complex of innocuous bio-protectors

Stable suspensions of metal oxide nanoparticles (Me-NPs) obtained by laser ablation of 99.99% pure copper, zinc or lead under a layer of deionized water were used separately, in three binary combinations and a triple combination in two independent experiments on rats. In one of the experiments the rats were instilled with Me-NPs intratracheally (i.t.) (for performing a broncho-alveolar lavage in 24h to estimate the cytological and biochemical indices of the response of the lower airways), while in the other, Me-NPs were repeatedly injected intraperitoneally (i.p.) 18 times during 6 weeks (for estimating the accumulation of corresponding metals in the blood and their excretion with urine and feces and for assessing subchronic intoxication by a large number of functional and morphological indices). Mathematical description of the results from both experiments with the help of the Response Surface Methodology has shown that, as well as in the case of any other binary toxic combinations previously investigated by us, the response of the organism to a simultaneous exposure to any two of the Me-NPs under study is characterized by complex interactions between all possible types of combined toxicity (additivity, subadditivity or superadditivity of unidirectional action and different variants of opposite effects) depending on which effect it is estimated for as well as on the levels of the effect and dose. With any third Me-NP species acting in the background, the type of combined toxicity displayed by the other two may change significantly (as in the earlier described case of a triple combination of soluble metal salts). It is shown that various harmful effects produced by CuO-NP+ZnO-NP+PbO-NP combination may be substantially attenuated by giving rats per os a complex of innocuous bioactive substances theoretically expected to provide a protective integral and/or metal-specific effect during one month before i.t. instillation or during the entire period of i.p. injections.

Toxicodynamic and Toxicokinetic Descriptors of Combined Chromium (VI) and Nickel Toxicity

After repeated intraperitoneal injections of nickel and chromium (VI) salts to rats, we found, and confirmed by mathematical modeling, that their combined subchronic toxicity can either be of additive type or depart from it (predominantly toward subadditivity) depending on the effect assessed. Against the background of moderate systemic toxicity, the combination under study proved to possess a marked additive genotoxicity assessed by means of the random amplification of polymorphic DNA test. We also demonstrated that chromium and nickel reciprocally influenced the retention of these metals in some organs (especially in the spleen) but not their urinary excretion in this study.

An Approach to Characterizing the Type of Combined Environmental Toxicity Based on Epidemiologically Assessed Exposure-Response Relationships

Using a dataset obtained in an earlier published epidemiological study that revealed the dependence of the probability of subclinical kidney damage in 260 children on the concentration of lead and cadmium in their urine, we have tested some methodological approaches to assessing the type of combined nephrotoxicity produced by these two metals. We have found that the environmentally caused damage to kidneys in children from lead and cadmium is less than additive (manifestation of toxicological antagonism). Given the subadditivity (antagonism) of the damaging effect of lead and cadmium on kidneys as demonstrated on the basis of epidemiological data, we believe that the summation of corresponding risks is a sufficiently conservative principle creating an additional margin of safety and limiting the uncertainty of risk assessment on the whole. Of theoretical interest is the demonstrated consistency of this assessment of the type of combined toxicity irrespective of whether it is carried out on the basis of the paradigm of effect additivity or dose additivity. This enables us to speak in favor of considering the so-called Bliss independence and Loewe additivity as complementary simplified models of the same fairly complex process rather than essentially different biological phenomena. The example studied suggests that approaches to the analysis of epidemiological data for the purpose of assessing combined risk should be tested using each of these models rather than restricting it to one of them based on an a priori choice.

Identification of combined action types in experiments with two toxicants: a response surface linear model with a cross term

Abstract Within the framework of the response surface linear model with a cross term, i.e. a model of the type Y(x1, x2) = b0 + b1x1 + b2x2 + b12x1x2 (hyperbolic paraboloid), a complete solution of identification of combined action types of two toxicants x1 and x2 is presented. It is shown that the type of combined effect in this model is determined by two factors: the direction in which the toxicants act (unidirectional or oppositely directed), and the position of the saddle point S of a hyperbolic paraboloid. For unidirectional actions of toxicants, already-known ways to identify the type of combined effect (including a shape of the isobole: concave-up or concave-down) provided identical and unambiguous answers regarding the type of combined effect (antagonism or synergism). For oppositely directed actions of toxicants, the shape of the isobole (concave-up or concave-down) did not allow us to determine the type of combined action type unambiguously. We show that in both cases (unidirectional or oppositely directed actions of toxicants) the signs of the model coefficients b1, b2 and b12, in conjunction with the coordinates of the saddle point S help unambiguously identify the type of combined action by comparing the observed effect with the zero interaction response surface. An atlas of all possibly combined action types for two toxicants for the hyperbolic paraboloid model was created. Applications of the developed formalism to experimental data are provided.

Experimental study and mathematical modeling of toxic metals combined action as a scientific foundation for occupational and environmental health risk assessment. A summary of results obtained by the Ekaterinburg research team (Russia)

Graphical abstract

Experimental Research into Metallic and Metal Oxide Nanoparticle Toxicity In Vivo

We studied purposefully produced silver, gold, iron oxide, copper oxide, nickel oxide, manganese oxide, lead oxide, and zinc oxide nanoparticles using two experimental models: (a) a single intratracheal (IT) instillation in low doses 24 h before the bronchoalveolar lavage to obtain a fluid for cytological and biochemical assessment; (b) repeated intraperitoneal (IP) injections during 6–7 weeks in non-lethal doses to assess the thus induced subchronic intoxication by a lot of functional and morphological indices and by the distribution and elimination of respective nanoparticles. Along with assessing the toxicity of these metallic nanoparticles (Me-NPs) acting separately, we also studied the same effects of some practically relevant Me-NP combinations. Besides, we carried out a 10-month inhalation experiment with an iron oxide (Fe2O3) nano-aerosol. We demonstrated that Me-NPs are much more noxious as compared with their fine micrometric counterparts although physiological mechanisms of their elimination from lungs proved highly active. At the same time, the in situ cytotoxicity, organ-systemic toxicity and in vivo genotoxicity of Me-NPs having a given geometry strongly depends on their chemical nature as well as on the specific mechanisms of action characteristic of a given metal. Even though being water-insoluble, Me-NPs are significantly solubilized in some biological milieus, and this process plays an important part in their biokinetics in vivo. In toto, Me-NPs are one of the most dangerous occupational and environmental hazards due to their cytotoxicity and genotoxicity, and therefore standards or recommended values of presumably safe Me-NP concentrations in the workplace and ambient air should be significantly lower as compared with those established for their micrometric counterparts. At the same time, the toxicity and even genotoxicity of Me-NPs can be significantly attenuated by background or preliminary administration of adequately composed combinations of some bioactive agents in innocuous doses.