Research Methods
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Research Methods
UDC 613.632.4-07:616.12-008.1-072.7
Professors I. M. Trakhtenberg, I. V. Sanotsky, and V. A. Tychinin; Candidates of Medical Sciences G. E. Verich, N. S. Grodetskaya, and A. M. Lukaneva
Experimental Assessment of the State of the Cardiovascular System as a Criterion for Establishing Hygienic Standards for Harmful Substances
Kiev Research Institute of Occupational Hygiene and Occupational Diseases; Institute of Occupational Hygiene and Occupational Diseases, Academy of Medical Sciences of the USSR; and All-Union Research Institute of Hygiene and Toxicology of Pesticides, Polymers, and Plastics, Kiev
One of the major shortcomings in the experimental assessment of the effects of harmful substances on the cardiovascular system for the purpose of establishing hygienic standards is the absence of standardized guidelines governing the relevant principles and methodological techniques, as well as a lack of synthesis and systematization of the data accumulated.
Over the years, hygienists and toxicologists have gained some experience in methodological approaches to the experimental study of the effects of harmful substances on the cardiovascular system. We may cite the work of V. A. Ishtykov and S. V. Speransky, who propose a method for determining the degree of selectivity with which harmful chemicals act on the cardiovascular system. In our view, however, the authors’ recommendations, as presented, cannot be put into practice for several reasons. First, it is hardly possible to propose a universal classification on the basis of rather arbitrarily selected reference compounds and doses. Second, the advisability of confining the assessment to ECG is open to question. Moreover, according to our data, the waves and intervals of the ECG cannot be distinguished in white mice because of their high heart rate. The recommendation to use a pituitrin challenge as a universal test is also debatable, since it is known that detecting latent changes in the functional activity of the heart and blood vessels requires a variety of challenge tests that activate different components of cardiovascular regulation.
A number of approaches have been proposed for the rapid detection of the effects of harmful industrial substances on the cardiovascular system. Thus, L. P. Sgibneva recommends using the following measures and tests: ECG; determination of capillary fragility and blood flow velocity in white mice, the duration of cardiac automaticity, the strength of the muscular heart wall, and systolic pressure; as well as recording the activity of the frog heart,
isolated by Straub’s method. The idea of introducing rapid methods for detecting the cardiotoxic effects of harmful substances into toxicological research practice is undoubtedly worthy of attention. Unfortunately, however, the recommendations in question have the same shortcomings as those proposed by V. A. Ishtykov and S. V. Speransky. Moreover, the advisability and validity of experiments using an isolated frog heart, a crude mechanical method for determining the strength of the muscular wall of the mouse heart, and an imperfect method for measuring blood flow velocity from the time taken for a dye injected into the retro-orbital sinus to spread, among others, are open to question.
This paper presents the rationale for, and a scheme for, studying harmful chemical compounds at successive stages of toxicological experimentation.
The cardiovascular system should be investigated alongside other organs and systems at all stages of the toxicological assessment of chemical compounds: when establishing thresholds for acute and chronic effects, identifying delayed effects of exposure, developing experimental treatments and preventive measures, and conducting clinical and hygienic assessments of populations exposed to the chemical compound under study.
The proposed scheme for toxicological experimentation can be used not only to establish hygienic exposure limits for industrial chemical compounds, but also to study the effects of pesticides, polymers, and other materials.
Particular attention should be paid to the cardiovascular system when a compound has a pronounced capacity to accumulate in the body, or when large population groups are exposed to it in occupational or domestic settings.
When determining the threshold for acute effects and the specificity of the observed cardiovascular changes, electrocardiographic data obtained at rest and after functional challenges (orthostatic, pharmacological, and others) should be analyzed alongside assessments of other systems and organs (the nervous system, liver, kidneys, etc.). The analysis should take account of heart rate, ECG configuration, the $P$–$Q$ and $Q$–$T$ intervals, and the voltage of the $R$ and $T$ waves. A comprehensive assessment of myocardial status requires investigation of the biochemical substrate of the observed electrophysiological changes in the myocardium, including the activity of isoenzymes of lactate dehydrogenase and creatine phosphatase, as well as morphometric parameters, including the thickness of myocardial muscle bundles and nuclear-to-cytoplasmic ratios in myofibrils. An overall assessment of peripheral vascular tone may be limited to the measurement of arterial blood pressure.
If the chemical compounds under study act selectively on the cardiovascular system ($Z_{\mathrm{ph}}>1$), indices of its condition are used in chronic experiments as pathogenetic criteria for establishing $\mathrm{Lim}_{\mathrm{ch}}$. Functional data should then include evaluation of the ECG at rest and after a battery of functional challenges, measurement of arterial blood pressure, and, where indicated, integral rheography.
These methods make it possible to assess heart rate, ECG configuration, the duration of the $P$–$Q$ and $Q$–$T$ intervals, the voltage of the $R$ and $T$ waves, cardiac output and stroke volume, total peripheral vascular resistance, and systolic and diastolic arterial blood pressure. Functional challenge tests make it possible to detect latent changes in various components of the cardiovascular system and its regulation, and to assess the hygienic significance of the changes found. Biochemical assessment of myocardial energetics and of the state of oxidation–reduction processes in the myocardium is recommended. Morphological studies determine nuclear-to-cytoplasmic…
Scheme for Studying the Effects of Chemical Substances on the Cardiovascular System
| No. | Stage | Methods of investigation | Indicators characterizing the functional state of the cardiovascular system |
|---|---|---|---|
| 1 | Establishment of the acute-effect threshold | ECG in lead II and a chest lead | Heart rate, \(P–Q\) and \(Q–T\), configuration and voltage of the \(R\) and \(T\) waves* |
| 1 | Establishment of the acute-effect threshold | Functional (stress) test | Heart rate (primary indicator), configuration and voltage of ECG waves |
| 1 | Establishment of the acute-effect threshold | Measurement of arterial blood pressure | Blood pressure, cardiac output, stroke volume, total peripheral resistance |
| 1 | Establishment of the acute-effect threshold | Determination of cardiac output and stroke volume (desirable) | |
| 1 | Establishment of the acute-effect threshold | If feasible, biochemical methods (determination of lactate dehydrogenase and creatine phosphatase in blood serum) and morphological methods with myocardial morphometry (general staining, azure II–eosin staining, morphometric assessment of nuclear-to-cytoplasmic ratios and myofibril bundle thickness) | |
| 2 | Establishment of the chronic-effect threshold | ECG in lead II and a chest lead, measurement of arterial blood pressure and, if feasible, integral rheography | Same as in item 1, and blood pressure; cardiac output, blood pressure, heart rate, total peripheral resistance |
| 2 | Establishment of the chronic-effect threshold | Orthostatic test | Same as in item 1; with integral rheography: cardiac output, blood pressure, total peripheral resistance, stroke volume |
| 2 | Establishment of the chronic-effect threshold | Physical exercise test | Same |
| 2 | Establishment of the chronic-effect threshold | Pharmacological test | Same |
| 2 | Establishment of the chronic-effect threshold | Biochemical methods: determination of lactate dehydrogenase and creatine phosphatase in blood serum; if feasible, determination of high-energy compounds (ATP, ADP, AMP), glycolysis and oxidation–reduction enzymes (cytochrome oxidase and succinate dehydrogenase) | |
| 2 | Establishment of the chronic-effect threshold | Morphological methods as used to establish the acute-effect threshold, and morphological assessment of the myocardium, aorta and vessels | |
| 3 | Study of the mechanism of action | Functional methods as in item 2; pharmacological tests with substances whose mechanisms of action are known | Cardiac output, stroke volume, blood pressure, heart rate, total peripheral resistance, \(P–Q\) and \(Q–T\), configuration and voltage of ECG waves |
| 3 | Study of the mechanism of action | Perfusion of the sinocarotid zone | |
| 3 | Study of the mechanism of action | Determination of the contractile properties of vascular smooth muscle | Frequency and amplitude of vascular strip contractions, nature of the response |
| 3 | Study of the mechanism of action | Biochemical methods as in item 2 | |
| 3 | Study of the mechanism of action | Morphological methods as in item 2, and determination of catecholamines by the Falck method, vascular elastic tissue and argyrophilic fibers; micrometry of peripheral vessels | |
| 4 | Prediction of long-term consequences | Investigations at long intervals, with assessment of natural aging | |
| 4 | Prediction of long-term consequences | Functional methods as in item 2 | Same as in item 2 |
| 4 | Prediction of long-term consequences | Biochemical methods as in item 2, and investigation of lipid metabolism | |
| 4 | Prediction of long-term consequences | Morphological methods: morphometric assessment of the myocardium, aorta and muscular-elastic vessels |
Continued
| No. | Stage | Methods of investigation | Indicators characterizing the functional state of the cardiovascular system |
|---|---|---|---|
| 5 | Experimental therapy | Modeling one form of cardiovascular pathology (coronary insufficiency, arterial hypertension, atherosclerosis, or adrenaline-induced myocarditis). Evaluation of the therapeutic effect of the pharmacological agents used. Functional, biochemical, and morphological methods, as in item 2. | The same as in item 2. |
* In rabbits, the configuration and voltage of the \(P\) and \(S\) waves should be determined.
…[[unclear: beginning of word]] relationships in the myofibrils, the ratio of connective tissue to myofibrils, and the thickness of myocardial muscle bundles; the thickness of the layers of the aortic wall and of the elastic membranes is measured, and the thickness of the layers of muscular-elastic vessels, the area of their tunica media, and their tone are assessed. The localization of the observed biochemical changes can be confirmed by quantitative histochemical studies.
When studying the delayed effects of toxic compounds on the heart and blood vessels for the purpose of establishing hygienic exposure limits at low exposure levels, it is recommended that the rate of progression of the “natural” aging processes of the heart and vessels be examined. The functional methods used and the indicators assessed are the same as in a chronic experiment. In biochemical studies, in addition to using the methods recommended above, particular attention should be paid to the state of lipoprotein metabolism and the composition of the connective tissue of large vessels. The latter appears to be important in the development of age-related changes in the cardiovascular system. Morphometric assessment is performed on the myocardium, aorta, and muscular-elastic vessels and, where indicated, on the vessels of the kidneys, brain, and heart.
Given the possibility that the chemical compounds under study may exert genetic and embryotoxic effects on the cardiovascular system of the fetus and offspring, examination of first-generation offspring for congenital abnormalities may be considered fully warranted.
One possible approach to studying the risk of delayed effects is to model specific forms of human cardiovascular pathology—atherosclerosis, hypertension, myocarditis, and coronary insufficiency—in animals. The results of the chronic experiment should be taken into account, as it establishes which components of the cardiovascular system are targeted. This is also very important in experimentally substantiating pathogenetically targeted treatments and preventive measures.
Subsequent comparison of the experimental data with the results of clinical-hygienic and pathological studies is highly desirable for the final validation of a model for experimentally predicting the delayed effects of chemical compounds on the cardiovascular system.
The scope of investigations undertaken to substantiate a therapeutic strategy corresponds to that of the chronic experiment. Two experimental approaches are advisable: administering therapeutic agents during or after exposure to determine their therapeutic effect, and administering them before exposure to determine their preventive effect.
Special attention should be paid to the selection of animals for studying the effects of chemical compounds on the cardiovascular system.
The generally accepted practice in hygienic standard-setting of using at least two species of laboratory animals in parallel should, in this case, be supplemented by the following considerations. When evaluating ECGs, rabbits and guinea pigs, whose ECGs more closely resemble those of humans, should be studied alongside rats. When determining the rate of development of natural aging processes, rats—the currently accepted gerontological model—are the principal animal species. A statistical group should include no fewer than 30 animals for each parameter. When modeling cardiovascular disease, rabbits should be used alongside rats. The use of laboratory animals that are currently rare, such as miniature pigs and monkeys, to study long-term effects is an important task for future research.
In conclusion, the need to further expand and deepen not only experimental but also clinical and clinical-statistical studies of the effects of toxic substances on the cardiovascular system deserves particular emphasis. Only by integrating animal experiments with in-depth observations in humans will it be possible to establish reliable hygienic standards and address the effective prevention of occupational injury to the heart and blood vessels.
References
Ishtykov, V. A., and Speransky, S. V. Scientific Transactions of the Moscow Research Institute of Hygiene, 1974, no. 21, pp. 50–54.
Sgibneva, L. P. On the Possibility of Accelerated Investigation of the Effects of Industrial Poisons on the Cardiovascular System in Toxicological Experiments. Candidate’s dissertation. Leningrad, 1967.
Received July 29, 1977.