Symposium on Clinical Toxicology for the Small Animal Practitioner
Adverse Drug Reactions Edward J. Gralla, V.M.D. *
Vast amounts of information deal with the broad subject of adverse drug reactions in the modern biomedical literature. Much of it concerns either human patients or studies in laboratory animals. That which can be classified as practical and immediately applicable to the practice of veterinary medicine will be found in other sections of this symposium. This article and the one that follows focus on the more basic insights that contribute to our understanding of clinical drug toxicity. These insights can be viewed as fundamental knowledge to any practitioner who must prescribe therapeutic use of drugs.
Toxicology Redefined Toxicology has traditionally referred to the diagnosis and management of poisonings resulting from the deliberate or inadvertent misuse of drugs or other toxic chemicals. In more recent times, the meaning has been extended to include a related but somewhat different area of medicine, that of unavoidable adverse reactions associated with the proper therapeutic application of drugs. This change has created differences that merit careful consideration. First is the role of preventive medicine. The most important principle of poison control has always been to avoid the offending agent and to prevent exposure or reexposure. This simple and reasonable solution is devalued in handling drug toxicity, for the offending drug may also be life-saving or life sustaining to the patient. A second major tactical change has been the value of detecting the toxic agent. In classical cases of poisoning, the analytical chemist has always been the clinician's closest colleague. The finding of a toxic chemical in the patient's tissues or body fluids confirmed the diagnosis. *Director of Toxicology, Mason Research Institute, Worcester, Massachusetts Veterinary Clinics of North America- Vol. 5, No.4, November, 1975
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This concept has lost any real value in the realm of drug toxicity for little is added to the clinician's diagnostic acumen to learn that a prescribed drug is appearing in the patient's blood or urine. More to the point is a whole battery of questions that must be asked and answered if the patient is to be protected from his medication. One should ask of a drug: What is the target organ(s)? What are the functional changes? What diagnostic measures are needed to detect the onset of toxic effects? Are the techniques sensitive, or must the damage be advanced before it is detectable? Is the damage reversible, i.e., do the organ tissues regenerate and effectively repair following drug withdrawal, or is the change permanent? Finally, a question that hopefully is asked before a prescription is written, what are the benefits to the patient compared with the risks of drug injury? All these questions are basically clinical in nature and relate to chemically-induced organ malfunction. Therefore our system for considering adverse drug reactions will be from the viewpoint of specific organ effects.
HEPATOTOXICITY The liver can be singled out as the organ most susceptible and most often damaged by chemical insults. Through it passes all the potentially toxic drugs and other chemicals absorbed from the gut. The major share of drug metabolism occurs here and so it follows that there is a maximal concentration of drug molecules undergoing inactivation, or sometimes activation from a nontoxic to a toxic state. Because of this exposure and susceptibility to toxicity, more is known about adverse chemical effects on the liver than probably any other organ. Inevitably, such a large body of knowledge becomes divided into more manageable subgroupings. Zimmerman10 has recommended the classification of hepatotoxins into direct, indirect, and unclassified groups. This suggestion has generally become accepted and will be followed in this report. Direct Hepatotoxins
The classification of hepatotoxins into direct or indirect is based on the following distinguishing characteristics: the number of species that can be affected; the incidence of toxicity after exposure; the time interval between exposure to the toxin and the onset of toxicity; and the intrahepatic lesion. Several features may be considered. The most impressive is the universally devastating manner in which the direct toxins act. These chemical sledgehammers act indiscriminately in both sexes of all species. As we shall see, indirect hepatotoxins require refinements like nontoxic sensitizing exposures and latency periods; direct toxins act
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quickly the first time every time. The hepatic cells are attacked in a forceful manner, showing fat accumulation soon after exposure which progresses to typical hepatocellular necrosis accompanied by nuclear pyknosis and disintegration, cytoplasmic swelling and vacuolation leading to eventual cell loss and fibrosis, or cirrhosis. Signs of overt destruction extend to the subcellular structures including the endoplasmic ret.icula, mitochondria, and lysosomes. The intrahepatic distribution is also a characteristic feature, being zonal and spreading peripherally from the central vein of the hepatic lobule. Cells in this affected area are reported to be highest in the enzymes which metabolize drugs and other foreign molecules. The importance of this observation became clear with the discovery that at least one hepatotoxin, carbon tetrachloride, becomes activated when it is metabolically converted to a toxin, the free radical CCL3 +. * The oxidizing properties of this agent attack the cell membrane lipids by perioxidation, rendering the cell wall impervious to the triglycerides that are constantly being synthesized within the cell. Thus they accumulate and cause the fatty liver condition previously mentioned. A liver damaged in this manner loses its functional capabilities, such as bilirubin conjugatio-n. This leads to a true classic hepatitis. Serum bilirubin accumulates causing jaundice; hepatic transaminase enzymes lost from the damaged cells cause an elevation in the serum activity. Alkaline phosphatase excretion is impaired and so this enzyme also builds up in the serum. Because they are universally effective, toxins of this type are quickly and easily recognized. Animal exposure is usually under restricted or accidental circumstances. Indirect Hepatotoxins
Whereas the direct hepatotoxins operate in a shotgun-like manner causing a widespread destruction, the indirect hepatotoxins are more selective in their action. Part of this subtlety arises from the fact that immune mechanisms seem to be involved. Consequently, an indirect hepatotoxic effect requires one or more sensitizing doses which are otherwise innocuous. It is the unpredictable second or third or later treatment that is the damaging event, causing post-hepatic cholestasis. The mechanism for this has been circumstantially identified. Histologically, the major bile ducts are dilated by bile plugs and surrounded by eosinophile infiltrations. In the early stages of this disease the parenchymal cells are undamaged and ultrastructural units are intact and *It follows then that the toxicity of such an agent will be dependent on both the amount, or total body burden, of a drug and the rate at which the conversion to a toxin takes place. Since the hepatic drug metabolizing systems are sensitive to chemical stimuli, both stimulating and depressing, the toxic potency of a chemical can vary from animal to animal exclusive of dosage. This important concept will be dealt with in the subsequent article on Drug Interaction.
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will remain unaffected provided the offending chemical is withdrawn. The only lesion found seems to explain the toxic response. Bile duct canaliculi are swollen and distended and apparently obstruct the small bile ducts thus preventing normal bile flow. The drugs most often reported to cause this response are the anabolic steroids, such as methyltestosterone, and long-term administration of phenothiazine tranquilizers, such as chlorpromazine. Literature references that describe indirect toxicity are confined to a small proportion of all exposed patients. Moreover, despite repeated attempts the condition has not been reproduced in animals. This type of hepatotoxicity is unreported in veterinary medicine. Perhaps it is the species restriction; perhaps it is because the population of veterinary patients receiving these drugs is relatively smaller and treatment periods are shorter; or perhaps the condition does occur on a small scale and is unrecognized.
Unclassified This is more than a catch-all category since it includes many of the more recently discovered hepatotoxins that cannot be simply classified as direct or indirect. A case in point is the volatile anesthesic, halothane, that causes a type of hepatotoxicity with features of both the direct and indirect variety. The recognition of halothane hepatitis was troublesome and elusive for many years because of the inconsistent pattern in which it appeared. This drug was u.sed successfully in surgical patients with no suggestion of toxicity appearing until time passed and a considerable number of patients had been reexposed. It was then realized that classic hepatitis with jaundice and serum transaminase elevations occurred in some patients after one or more uneventful exposures. Thus this agent appeared to require sensitization and therefore, it was a typical indirect hepatotoxin. However, the analogy failed when the organ lesion was studied and found to consist of the direct type, zonal hepatocellular necrosis. Moreover, mitochondrial membrane damage and disruption were found by electron microscopy. While all clinical reports of halothane hepatitis have been confined to the human literature, it serves as a classic example of unexpected and incipient drug adversity and provides an objective lesson for practitioners of veterinary mediCine. Another drug-related hepatitis reported to be associated with a specific physiological state is tetracycline-induced, fatty liver of pregnancy. A causal relationship between this condition and tetracycline administration has been difficult to prove and is still disputed. The main point of contention centers around the fact that identical clinical and morphological lesions, jaundice, and fine fatty vacuolization of hepatocytes, have occurred in susceptible women in the last trimester of pregnancy who have not received any medication. In fact, the disease
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was first reported in 1940 before the tetracyclines were discovered. Nevertheless, an increased incidence in patients receiving tetracyclines in late pregnancy has been reported from numerous sources. The use of this class of antibiotics in pregnancy has been for the most part discontinued because of a totally unrelated problem. These agents chelate, or bind, to the calcium in calcified tissues and thus cause yellow tooth discoloration in the offspring of treated mothers. The question of maternal hepatic changes will probably remain unresolved.
NEPHROTOXICITY Prerenal Events Drug-related acute renal failure, classicially characterized by oliguria, is usually the result of a prerenal toxic effect, for example, a hemolytic crisis leading to an excessive load of hemoglobin being presented to the kidneys. A similar condition may also be induced by intramuscular injection of drugs that damage muscle tissue, such as glycerol, wherein myoglobin is released and becomes the destructive agent. Another drug-induced indirect systemic effect is urate nephropathy following anti-cancer drug therapy. The destruction of massive numbers of neoplastic cells releases nucleic acids that are metabolized through normal catabolic pathways to uric acid and its salts. These products have a limited solubility in body fluids which is exceeded and a precipitation of solid crystals within the renal tubules follows. Plugging of the lumina and mechanical damage to the lining cells are the result. A notable example of a countermeasure to a toxic effect has evolved around this problem for it has become a routine practice in human cancer chemotherapy to co-administer allopurinol, a xanthine oxidase inhibitor, which blocks the conversion of nucleic acids to uric acid and thus protects the kidney.
Effects on Glomerulus The glomerulus is rarely subjected to toxic damage despite a high degree of exposure to unbound and therefore, highly active, low molecular weight drug molecules that cross the glomerular membranes. This is fortunate, for the glomerulus, unlike the tubules, is not known to be regenerative. This protection probably arises from the general inert state of glomerular substructures that appear to lack the sophisticated enzyme systems found elsewhere in this organ. Furthermore, both foreign and endogenous molecules pass rapidly from plasma to filtrate so the time of exposure is short. This structure is not completely spared, however. At least one drug has been implicated in causing a specific glomerular toxiCity at prescribed clinical dosages. Dpenicillamine has received scattered reports as a cause of a glomerular nephrosis.
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Effects on Tubules
Down in the lower renal structures, the tubules, where complicated biochemical processes cause drug and water molecules to ebb and flow, the list of toxic substances begins to grow. In the tubules molecular size seems to have a bearing on the structure affected. Heavy metals, such as lead, mercury, gold, and cadmium, damage both the proximal and distal tubules. On the other hand, organic molecules damage only the proximal tubules, and so a list of drugs headed by the antibiotics polymyxin, neomycin, bacitracin, kanamycin, and the tetracyclines are known to cause lesions in these structures, such as cellular necrosis and sloughing, swollen tubules, and intraluminal protein leakage. The type of tubular damage is reflected by functional changes. Resorption of compounds such as glucose, amino acids, and phosphates takes place in the proximal tubules: A typical response to lesions in these structures is glycosuria, aminoaciduria, hyperphosphaturia, and hypophosphatemia. The distal tubutes serve the function of ion exchange and buffering effects, so that damage to these structures is reflected by hyperkaluria and renal acidosis. A more unusual biochemical type of distal tubular lesion has appeared with the advent of lithium treatment in psychiatry. Patients receiving doses causing high serum levels of this element developed a classic diabetes insipidus with polydipsia, polyuria, and a loss of urinary concentrating ability that was unresponsive to injections of antidiuretic hormone. The exact mechanism for this effect is unclear but since lithium is chemically similar to sodium, it suggests the possibility of a competitive antagonism between these two elements for excretory or resorptive mechanisms. Other Effects
At least one compound, methoxyflurane, has the potential for either organic or inorganic toxic effects and this fact has surfaced in clinical reports. The parent molecule of this anesthetic agent can be metabolized in vivo to oxalate, or fluoride, or both; both are nephrotoxins with different mechanisms of action. Excess oxalate causes crystalluria in the proximal tubules. Fluoride nephrotoxicity is more of a biochemical lesion characterized by glycosuria and polyuria without visible lesions. The intrarenal concentrations of the metabolite that accumulate depends upon the relative rate and activity level of the respective enzyme system. Having previously identified the tetracyclines as nephrotoxic agents several additional points should also be mentioned. First is that these drugs deteriorate slowly during storage to form two potent nephrotoxic by-products, anhydro-4-tetracycline and epianhydrotetracycline. Several fatal clinical reactions reported were traced to this effect, permanent reminders of the wisdom in discarding outdated drugs. A second effect tetracyclines have is to cause prerenal uremia, which can be
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serious if it is superimposed on preexisting uremia. This relates to the effect of tetracyclines on blocking protein synthesis. Presumably this prevents amino acids from entering the normal synthetic pathways and instead they are metabolized into nitrogenous compounds such as urea. The liver and kidneys are key excretory organs for removing foreign and toxic chemicals, with a measure of protective interdependence. The results of upsetting this balance are well illustrated by an outbreak of nephrotoxicity caused by the radicicontrast media bunaminodyl and iopanoic acid. These agents are removed by the biliary system and, for this reason, they were developed specifically to outline the gallbladder and bile ducts. As long as the biliary tree was patent no toxic reactions occurred. However, cholestasis caused the m~or burden of drug excretion to fall upon the kidney and cause severe tubular damage in this type of patient. This toxicity mechanism was belatedly demonstrated in dogs with ligated bile ducts. While overt cellular destruction and/or biochemical disruption of critical enzyme systems are primary mechanisms for chemically induced nephrotoxicity, a third system for chemical interference can be the opposite, that of cellular proliferation. This effect caused by the anticancer drug mitomycin C was discovered in the laboratory in time to prevent a similar occurrence in patients. Mice treated with this chemical at clinically subtoxic doses developed hydronephrosis. The cause was traced to a dose-related epithelial proliferation with papilla formation in the renal pelvis which effectively obstructed urine flow to the ureter. The resulting back pressure caused a dilatation and degeneration of the collecting tubules.
HEMATOPOIETIC TOXICITY The toxic effects in this category are manifested clinically as reductions in the level of circulating blood cells, usually erythrocytes, neutrophils or platelets, and occasionally lymphocytes. These changes come about as a result of either a direct action on the mature cell that has entered the blood stream or a depressing effect on the organs that generate replacement cells, the bone marrow, or lymphoid tissues. The type of toxic mechanism involved can lead to differences in the clinical response which are diagnostically important. First is the time lapse following the toxic insult before recognizable changes take place. A direct cellular toxin will destroy the cell quickly, circulating levels will fall rapidly, the number of immature replacement cells will increase and hyperbilirubinemia will occur. When bone marrow toxicity is involved and cell production is halted then the circulating levels will not diminish until normal attrition causes a significant reduction in the circulation, without the appearance of immature replacement cells or elevated bilirubin levels. The short life of a neutrophil, being four or
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five days under normal conditions, means that a drug toxicity which shuts off myelogenesis will not become evident for 48 hours, or when circulating values fall to half the initial levels. A similar erythrocytic effect would not be revealed for a longer period of time since the halflife of this cell is approximately 100 days. With this as a background we shall consider specific examples. Hemolytic Anemias
Anemias can be caused by an agent attacking either the erythrocytic cell membrane or the biochemical interior. Lead and copper are good examples of membrane toxins, since like most bivalent heavy metals, these elements accumulate at the cell surface.* The affected cell loses potassium and at the same time becomes more fragile, lysing in osmotic solutions that would have no effect on healthy cells. Cellular components are also attacked by these agents but presumably in somewhat different ways since copper toxicity is indicated by Heinz body formation, whereas lead causes a basophilic stippling especially in reticulocytes. An organic compound that acts in a manner similar to copper is diphenylhydrazine, a discarded hypotensive drug only rarely used and then to clinically treat polycythemia vera. These direct-acting hemolytic agents appear to damage or alter the red blood cells so that they are prematurely removed from the circulation by the spleen and destroyed. The mature erythrocyte is a metabolically active cell with a limited supply of enzymes. One of these, glucose-6-phosphate dehydrogenase (G-6-PD), is subject to a chemical inhibition that becomes a toxic reaction under predisposing circumstances. All of the cases so far reported have involved humans from specific ethnic backgrounds, either blacks or individuals whose ancestry can be traced to countries bordering the Mediterranean Sea. The red blood cells of these patients have a limited amount of G-6-PD. This enzyme is part of an important biochemical system that reduces or detoxifies the small amount of methemoglobin normally formed in the red blood cell as hemoglobin is oxidized. Several drugs, such as the antimalarial primiquine and the antibacterial nitrofurans and one natural food, fava beans, are active inhibitors of G-6-PD. The combination of minimal levels of enzyme and treatment by an active inhibitor causes the older erythrocytes with higher levels of methemoglobin to be destroyed, presumably by the spleen, leading to a drug-induced hemolytic anemia. This toxicity has been confined to congenitally predisposed humans; therefore, the condition is unknown in other ethnic groups, and seems to be far from the realm of veterinary medicine. It does, however, serve to illustrate a toxicity caused by a drug-gene interaction. *That they accumulate at the cell surface must be remembered when blood samples are taken for analysis of heavy metals. Whole blood is required, never serum or plasma.
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Immune Reactions
Immune mechanisms are important in drug-induced anemias, including the hemolytic variety. One of the most common involving the erythrocyte is the so-called hapten or penicillin reaction. In this example the drug acts as hapten following conjugation with a body protein to form an antigen. In susceptible patients, this stimulates the formation of an IgG type antibody that coats the red blood cell.* Subsequent treatment with the antigenic drug causes an antigenantibody reaction that destroys the erythrocytes. In a second, so-called "innocent bystander" autoimmune reaction, certain drugs, such as phenacetin, chlorpromazine, and some of the sulfonamides, have been reported to induce the formation of a humoral antibody. Additional drug treatments provide a source of antigen and the antibody-antigen combination becomes loosely attached to the red cell surface activating complement and causing cell lysis. Because the attachment to the erythrocyte is tenuous, the antibody is released when the cell is destroyed and becomes free to react with other antigens. This means that relatively small, amounts of antibody can be recycled to cause a disproportionately greater degree of destruction. The third hemolytic mechanism has been labeled the "methyl dopa" reaction after the only drug reported to be involved. Since these patients are Coombs' positive, the most accepted theory explaining this reaction is that the drug somehow alters the erythrocyte rendering it antigenic and causing the production of autoantibodies. Only the immune system has been identified in leading to an active depletion of circulating leucocytes and thrombocytes in clinical cases involving humans. The more diverse and sophisticated mechanisms that destroy erythrocytes are as yet undiscovered. The main body of evidence supporting the hypothesis for leukoagglutinins has come from studies where cells and serum from patients and unaffected humans have been intermixed and the results followed. Usually the combination of normal cells and sensitized serum will produce no response until the offending drug is added, and then a visible agglutination occurs. The further addition of complement causes lysis. Another line of evidence evolves from the lymphocyte transformation test, which, incidentally, shows promise as a diagnostic procedure. In this system, lymphocytes from the patient are mixed with neutral serum containing the drug in question. A typical morphological change in the lymphocyte indicates an immune sensitivity. The drugs most frequently implicated in drug-induced agranulocytosis or thrombocytopenia have been amidopyrine, phenylbutazone, and chlorpromazine. *This is different from the classic penicillin hypersensitivity reaction that involves a serum IgM antibody.
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Effects on Bone Marrow
Agranulocytosis can also be the result of a specific block in myeloid maturation in the bone marrow. This has followed long-term treatment of human patients with high doses of chlorpromazine. In these cases, marrow smears were low in myeloblastic precursor cells. Stem cells were spared and normal function was confirmed by a restoration of neutrophil counts in two weeks following withdrawal of the offending drug. Both myeloid and erythroid cellulopoiesis can be destroyed by drugs, either temporarily or permanently. Three major examples of this toxic panleukopenia are cytotoxic drugs, benzene, and chloramphenicol. Cytotoxic drugs, including the antimetabolites, alkalating agents, and mitotic inhibitors, are primarily the anticancer agents and thus have been selected for one principle activity, potent cytotoxicity. Since they are designed to attack and destroy rapidly dividing cells, it is not surprising that the spectrum of activity includes the bone marrow. This is a well known effect that is detected early. Under most clinical conditions, drug treatments are administered cautiously and halted before the marrow is totally destroyed. At this stage recovery usually follows drug withdrawal. The most serious problem associated with this condition is the susceptibility of treated patients to infections. Therefore antibiotics should be instituted immediately at the earliest indication. Benzene exposure, either by ingestion, absorption of the liquid material through the skin, or inhaling the vapor, has developed a historically notorious reputation for causing complete marrow failure. The destruction included the stem cells and was inevitably fatal to the patient. Because of this. hazard the use of this compound has been severely restricted. This effect was clearly direct since it caused a l 00 per cent incidence following exposure to toxic levels and all species tested showed this response. The mechanism for chloramphenicol-induced aplastic anemia is more obscure. The overall reported incidence of bone marrow toxicity was extremely low with one reaction out of 100,000 patients and only in those individuals receiving high doses for long periods of time. However, the effect was permanent and irreversible so that mortality was high. The only change in blood chemistry that seemed to forewarn an impending toxic crisis was a rise in serum iron with iron binding capacity approaching saturation. Within the marrow, vacuolization of the normoblasts has been reported to be present in affected patients. A few studies have been done with the limited amount of clinical material that could be obtained. An inhibition of protein synthesis by the immature precursor cells has been reported as the primary lesion with a block in the generation of messenger RNA. In recent years, this antibiotic has been used very selectively and the incidence of aplastic anemia has been concomitantly reduced, so the exact nature of this disease will probably never be known.
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Functional Anemias Thus far this discussion of hematopoietic toxicity has considered conditions characterized by a reduction in the number of circulating cells. Another general category is the situation whereby the number of circulating cells is normal but they have been rendered functionally defective by a chemical. The most familiar illustration is methemoglobinemia, or the state of chemically oxidized ferrous ions within the hemoglobin molecule incapable of oxygen transport. At least one drug, the analgesic phenacetin, has been responsible for this toxicity. Another has been environmental nitrates in water supplies or from preserved meats. These are reduced to nitrites which are the toxic agents. The most potent chemicals for this purpose are the aniline dyes. These agents are extremely active so only a minimal exposure is required. For example, there are several reported outbreaks of methemoglobinemia in infants that were traced to the dye used for stamping laundry markings on their. diapers. A type of iron deficiency anemia has appeared in pigs fed cottonseed meal. The active toxin in this diet is gossypol which when extracted and purified causes microcytic, hypochromic anemia and death from pulmonary edema and cardiac failure, but interestingly, only in those species with simple stomachs. This agent has been shown to deplete liver iron and increase bile iron in rats, apparently the result of a chelating property. It has been known for many years that aspirin treatment prolongs clotting time. The exact mechanism for this effect was unknown until recently when it was shown to inhibit platelet aggregation, the first step in clot formation. The details of this experiment are worth considering. The addition of collagen to platelet-rich plasma is followed by a wave of aggregation measurable by appropriate laboratory techniques. The further addition of adenosine triphosphate (ATP) causes another wave of aggregation to occur. It is this second wave that is inhibited by aspirin. Furthermore subsequent studies have shown that this is a functional effect and apparently lasts for the life of the thrombocyte. Platelets transfused from treated donors that previously had received 600 mg of aspirin failed to correct the prolonged dotting time of thrombocytopenic patients until 36 hours after this treatment. At· this point, either the chemical effect was lost or new, unaffected platelets were regenerated and replaced those that were exposed. Spleen Toxicity Finally, in this discussion we shall consider spleen toxicity, a phenomenon reported from laboratory studies. Rats treated with the macromolecular polymers hydroxylamine, polyvinyl alcohol, or methylcellulose developed a normocytic, normochromic, Coombs' negative anemia with reticulocytosis, hyperplastic bone marrow, and spleno-
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megaly. The half-life of untreated red blood cells injected into treated animals was considerably shortened. Furthermore this response was absent in splenectomized rats. Taken collectively, this evidence pointed to a chemically induced, hypersplenism, or a spleen so hyperactive that it destroys erythrocytes long before they would normally expire. The opposite effect, spleen depression or destruction, has been produced in rats receiving single injections of an emulsion of ethyl palmitate, a fatty acid ester. Radioisotope studies have shown that this agent localizes in and destroys the splenic red pulp. One clinical implication of this effect was brought out in studies with rats infected with Hemobartonella muris. Under ordinary circumstances this is alatent infection. The active form, manifested by acute hemolytic anemia, can be precipitated by splenectomy or the chemical under discussion, ethyl palmitate.
GASTROINTESTINAL TOXICITY The gastrointestinal tract is a frequent target for toxins for several very good reasons. First, over and through the mucosal surface of this organ pass the molecules of orally administered drugs in high concentrations. Moreover, if the drug is absorbed and excreted in the bile, then the insult is compounded many times as the agent and/or its metabolites are recycled through the enterohepatic circulation. The second reason for the susceptibility of the gastrointestinal tract is the high rate of cell division constantly underway in this organ. Interference with Mucosal Regeneration
The germinal centers deep in the crypts constantly producing new columnar lining cells are especially vulnerable to cytotoxic drugs, such as the anticancer agents, designed to destroy tissues with rapidly dividing cell populations. For the newly formed cells to move from the crypts to the lining of the villi requires a fixed amount of time. The mature cells that line the villus are in a nondividing, drug-resistant state and they are likely to survive a drug insult and be lost in an uninterrupted pattern. The problem develops when they are not replaced by the drug-suppressed regeneration from the crypt. This simply means that there is a latent period or delayed response between treatment and toxic effect. The action, rather than a direct destruction of the lining mucosa, is one of nonreplacement of the cells which, as part of the normal cell turnover, move up the villar surface and are lost from the villar tip. The clinical effect is a dose-related diarrhea that ranges from loose stools to severe hemorrhagic discharges. As with any diarrhea, fluid and electrolyte losses are serious effects leading to dehydration, shock, renal failure, and death. Replacement therapy is essential and must be maintained until the mucosal lining has regenerated. During this type
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of toxic crisis the patient is also highly susceptible to systemic invasion by entering organisms, so that appropriate prophylactic treatment with antibiotics is in order. Ulcerations
A second m~or type of adverse gastrointestinal effect is drug-induced ulceration of either the upper or lower tract. The anti-inflammatory drugs, such as corticosteroids, aspirin, phenylbutazone, and indomethacin, are the most common causes of drug-induced ulcer in the upper levels of the tract or the stomach and small intestine. Attempts have been made to define the mechanism for this toxicity. Several facts have emerged but no overall answers. The combination of drug and bile appears to be important since in one study no lesions appeared in phenylbutazone-treated, bile duct ligated rats. Furthermore bile collected from phenylbutazone-treated rats was a potent ulcerogenic agent in normal rats. Other lines of investigation have suggested that the permeability of the so-called "mucosal barrier" is altered to permit hydrogen ions to penetrate and damage the submucosal structures. In contrast to the diffuse nature of the cytotoxic-drug type of damage, these lesions are for the most part focal but deeply erosive. Mortality usually results from peritonitis which follows intestinal perforation. Similar ulcers have appeared in the small bowel of patients receiving enteric-coated potassium chloride tablets as a precaution against diuretic-induced hypokalemia. The same type of lesion was experimentally reproduced in dogs and monkeys treated with the potassium salt alone. The exact reason for this response has never been clearly defined. However, the lesions have been reported to include histological changes such as fresh thrombi and fibrinoid necrosis which suggest that the toxic response is one of hemorrhagic infarctions provoked by the sudden release and absorption of high concentrations of potassium within a short segment of bowel. Colitis
A counter response to mucosal destruction, that of stimulated growth, has been reported in humans as pseudomembranous colitis associated with the use of lincomycin and its analogue clindamycin. The initial sign of toxicity has been profuse watery diarrhea in some cases severe enough to cause a profound hypotension. At laparotomy the colon was found to be flaccid and dilated. Both toxic megacolon and colonic perforations have been described. This condition is usually diagnosed by proctoscopic or colonoscopic examination where characteristic raised yellow-white plaques are recognized. The process is reversible following drug withdrawal. The mechanistic cause of this condition is uncertain but, because it is produced by two antibiotics effective against gram-negative anaerobes, especially the Bacteroides
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species, speculation has centered around a possible drug-bacteria interaction. Superinfections A drug-induced, bacteria-mediated toxicity would not be unprecedented and, in fact, has been given the name, drug-induced superinfection. Essentially, two main types have been recognized. One is an overgrowth of fungal organisms, such as the Candida species, the other an imbalance between enteric gram-positive and gram-negative organisms caused by an antibiotic with a narrow spectrum of activity. Symptoms of the latter, which can be experimentally produced within two or three days in rabbits treated with oral penicillin, are profuse watery diarrhea and gas distention of the large bowel. This condition is irreversible unless treated with antibiotics that have a broad or gramnegative spectrum. Pure cultures of gram-negative orgamsms are recovered from the colon of these rabbits. Malabsorption Finally, there is the condition of drug-induced malabsorptive states that has been reported in human cases or experimental animals. Two widely different drugs have had a remarkably similar clinical effect. The drugs are triparanol, an inhibitor of cholesterol biosynthesis, and neomycin, the gram-negative antibiotic. The clinical state is malabsorption of fats and sugars, similar to idiopathic steatorrhea in man, a similarity consistent at the cellular level where a common finding has been villus atrophy accompanied by an increased mitotic activity in the. cryptic mucosa. One notable difference relates to species sensitivity. A condition similar to that found in man has been produced in triparanoltreated rats. Attempts to reproduce the human condition experimentally with neomycin have been unsuccessful. It is beyond the scope of this discussion to present all the evidence that supports or refutes various hypotheses attempting to explain the mechanisms of action for these drugs. For our purposes, it is sufficient to recognize that another mechanism of gastrointestinal toxicity, that of malabsorption, has been amply demonstrated.
EMBRYO-FETAL TOXICITY The thalidomide tragedy of the 1960's focused worldwide attention on the realization that a drug or other chemical can seriously and permanently damage the developing embryo or·fetus without adversely affecting the mother. The most popular solution put forth in the aftermath of this disaster was one of therapeutic nihilism, of simply not treating during pregnancy. While superficially this might appear attractive and reasonable, in actual practice it becomes an oversimplification. The principal coun-
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terargument is based on a fact shared by both human and veterinary medicine, namely, that the developing embryo is most susceptible to toxic chemicals during early pregnancy when many organ systems are rapidly differentiating from primordial tissues. Most importantly this period occurs long before pregnancy is suspected. At issue is a question of whether or not critical therapeutic drugs should be administered to, or withheld from, a female animal because she might have conceived and might be in a critical stage of pregnancy. The more realistic alternative is to use drugs during pregnancy with intelligence, caution, and an understanding of the underlying problems. Stage-Specific Sensitivity To this end, several established principles can be cited. First, is stage-specific sensitivity, meaning that the period of rapid differentiation is virtually the only time that a developing organ can be adversely affected by chemical intervention. The fully differentiated organ is relatively insensitive to the previously toxic stimulus. The exact timing of these events for most pets and domestic animals is unknown, but in laboratory animals such as rodents it has been established that major structures such as heart, central nervous system, and the earliest limb formation begin to appear early in the post-implantation period, i.e., day eight or nine of a 21 day gestation period. On the other hand, cleft palate closure, one of the last major developmental events to take place, can be inhibited by an appropriate insult later in pregnancy, days 14 or 15 in rodents. There are many good practical examples, including thalidomide, that have amply demonstrated the principle of stage-specific sensitivity. An answer to the question "was the organ system which was malformed differentiating when the mother was treated with the drug in question?" can frequently strengthen or refute a suspicion of teratogenicity. Species Specificity Another principle never to be overlooked is that of species specificity. Many chemical teratogens are notoriously specific for a single species and extrapolation to others can be misleading. An interesting example is taken from studies in mice and rats, two species that appear to be close counterparts. Mice treated with corticosteroids during days 13 and 14 of pregnancy produce offspring with a high incidence of cleft palates. Rats are insensitive to this effect. On the other hand, the chemical meclizine hydrochloride, a popular antiemetic drug, given to pregnant rats during the same stage of pregnancy induces the same congenital defect, cleft palate; not so with mice that are resistant. Activation Studies with meclizine hydrochloride in rats and a second cleft palate inducing drug diphenylhydantoin in mice have illustrated another
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important concept that aids in our understanding both teratology and toxicology, that of activation versus inactivation of a toxin. Meclizine hydrochloride is converted to a potent teratogenic metabolite, norchlorcyclizine, by the liver. Concomitant treatment with a drug that depresses the rate of metabolic transformation reduces the number of malformed offspring. Pretreatment with a drug such as phenobarbital that stimulates this same system causes more of the toxin to be generated and consequently increases the number of pups with cleft palate. Diphenylhydantoin causes the same defect in mice for opposite reasons. Here the parent compound is the offender. Inhibited metabolism increases teratogenicity; stimulation reduces the effect. Pharmacological Effects Frequently a drug may adversely influence the fetus by way of its pharmacological properties. Two conditions must be met for this to happen. First, the molecules must cross the placenta, and most active drugs freely cross this barrier by simple diffusion. Second, the fetal organs must be developed and pharmacologically reactive. A familiar example to most practitioners is the depressing effect of maternally administered anesthetics on fetal respiration. Other inadvertent pharmacological effects to the human fetus have resulted from administration of other drugs such as hormones. Certain progestational steroids, having minor unsuspected androgenic properties, given to pregnant females to prevent abortion caused the female offspring to be born with masculine features. Iodides that were part of cough treatment preparations stimulated euthyroid goiters in the offspring. Hypoglycemic agents administered to the mother stimulated insulin secretion by both the maternal and fetal pancreas, and so several babies have reportedly been born showing hypoglycemic convulsions and might have died except for treatment with glucose infusions. Vasopressor substances such as the catecholamines, vasopressin, and ergot alkaloids have caused profound peripheral vasoconstriction leading to anoxia and gangrenous extremities in the fetus. Not surprisingly, the administration of therapeutic anticoagulants to the mother has caused widespread hemorrhaging in the offspring. Drugs can also cause toxic effects in the fetus that are common in adults. The aminoglycoside antibiotics, streptomycin, kanamycin, and gentamycin are well known ototoxins. These have been reported to cause permanent deafness in the children of mothers who were treated with these agents in late pregnancy. ACKNOWLEDGMENTS
The author thanks Drs. Margaret and David Hayden, Veterinary clinician and Veterinary pathologist, respectively, for critically reviewing this and the following article.
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REFERENCES I. Clegg, D.J.: Teratology. Ann. Rev. Pharmacol.,J1:409, 1971. 2. Croft, D. N.: The action of analgesic substances on the gastric mucosa.]. Pharm. Pharmac., 18:354, 1966. 3. Huguley, C. M., Lea,]. W., and Butts,]. A.: Adverse hematologic reactions to drugs. Prog. Hematol., 5:105, 1966. 4. Karnofsky, D. A.: Drugs as teratogens in animals and man. Ann. Rev. Pharmacol., 5:447, 1965. 5. Klatskin, G.: Mechanisms of toxic and drug induced hepatic injury. In Fink, B. R. (ed.): Toxicity of Anesthetics. Baltimore, Williams and Wilkins Co., 1968, Ch. 14. 6. Pisciotta, A. V.: Drug-induced leukopenia and aplastic anemia. Clin. Pharmacol. Therapeut., 12:13, 1971. 7. Robinson, J. W. L.: Experimental intestinal malabsorption states and their relation to clinical syndromes. Klin. Wschr., 50:173, 1972. 8. Schreiner, G. E.: Toxic nephropathy due to drugs, solvents and metals. Prog. Biochem. Pharmacol., 7:248, 1972. 9. Zbinden, G.: Experimental renal toxicity. In Rouiller, C., and Muller, A. F. (eds.): The Kidney: Morphology, Biochemistry, Physiology, Vol. 2. New York, Academic Press, 1969, Ch. 6. 10. Zimmerman, H.].: The spectrum of hepatotoxicity. Persp. Bioi. Med., 12:135, 1968.
Director of Toxicology Mason Research Institute Harvard Street Worcester, Massachusetts 01608
Adverse Drug Reactions Edward J. Gralla, V.M.D. *
Vast amounts of information deal with the broad subject of adverse drug reactions in the modern biomedical literature. Much of it concerns either human patients or studies in laboratory animals. That which can be classified as practical and immediately applicable to the practice of veterinary medicine will be found in other sections of this symposium. This article and the one that follows focus on the more basic insights that contribute to our understanding of clinical drug toxicity. These insights can be viewed as fundamental knowledge to any practitioner who must prescribe therapeutic use of drugs.
Toxicology Redefined Toxicology has traditionally referred to the diagnosis and management of poisonings resulting from the deliberate or inadvertent misuse of drugs or other toxic chemicals. In more recent times, the meaning has been extended to include a related but somewhat different area of medicine, that of unavoidable adverse reactions associated with the proper therapeutic application of drugs. This change has created differences that merit careful consideration. First is the role of preventive medicine. The most important principle of poison control has always been to avoid the offending agent and to prevent exposure or reexposure. This simple and reasonable solution is devalued in handling drug toxicity, for the offending drug may also be life-saving or life sustaining to the patient. A second major tactical change has been the value of detecting the toxic agent. In classical cases of poisoning, the analytical chemist has always been the clinician's closest colleague. The finding of a toxic chemical in the patient's tissues or body fluids confirmed the diagnosis. *Director of Toxicology, Mason Research Institute, Worcester, Massachusetts Veterinary Clinics of North America- Vol. 5, No.4, November, 1975
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This concept has lost any real value in the realm of drug toxicity for little is added to the clinician's diagnostic acumen to learn that a prescribed drug is appearing in the patient's blood or urine. More to the point is a whole battery of questions that must be asked and answered if the patient is to be protected from his medication. One should ask of a drug: What is the target organ(s)? What are the functional changes? What diagnostic measures are needed to detect the onset of toxic effects? Are the techniques sensitive, or must the damage be advanced before it is detectable? Is the damage reversible, i.e., do the organ tissues regenerate and effectively repair following drug withdrawal, or is the change permanent? Finally, a question that hopefully is asked before a prescription is written, what are the benefits to the patient compared with the risks of drug injury? All these questions are basically clinical in nature and relate to chemically-induced organ malfunction. Therefore our system for considering adverse drug reactions will be from the viewpoint of specific organ effects.
HEPATOTOXICITY The liver can be singled out as the organ most susceptible and most often damaged by chemical insults. Through it passes all the potentially toxic drugs and other chemicals absorbed from the gut. The major share of drug metabolism occurs here and so it follows that there is a maximal concentration of drug molecules undergoing inactivation, or sometimes activation from a nontoxic to a toxic state. Because of this exposure and susceptibility to toxicity, more is known about adverse chemical effects on the liver than probably any other organ. Inevitably, such a large body of knowledge becomes divided into more manageable subgroupings. Zimmerman10 has recommended the classification of hepatotoxins into direct, indirect, and unclassified groups. This suggestion has generally become accepted and will be followed in this report. Direct Hepatotoxins
The classification of hepatotoxins into direct or indirect is based on the following distinguishing characteristics: the number of species that can be affected; the incidence of toxicity after exposure; the time interval between exposure to the toxin and the onset of toxicity; and the intrahepatic lesion. Several features may be considered. The most impressive is the universally devastating manner in which the direct toxins act. These chemical sledgehammers act indiscriminately in both sexes of all species. As we shall see, indirect hepatotoxins require refinements like nontoxic sensitizing exposures and latency periods; direct toxins act
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quickly the first time every time. The hepatic cells are attacked in a forceful manner, showing fat accumulation soon after exposure which progresses to typical hepatocellular necrosis accompanied by nuclear pyknosis and disintegration, cytoplasmic swelling and vacuolation leading to eventual cell loss and fibrosis, or cirrhosis. Signs of overt destruction extend to the subcellular structures including the endoplasmic ret.icula, mitochondria, and lysosomes. The intrahepatic distribution is also a characteristic feature, being zonal and spreading peripherally from the central vein of the hepatic lobule. Cells in this affected area are reported to be highest in the enzymes which metabolize drugs and other foreign molecules. The importance of this observation became clear with the discovery that at least one hepatotoxin, carbon tetrachloride, becomes activated when it is metabolically converted to a toxin, the free radical CCL3 +. * The oxidizing properties of this agent attack the cell membrane lipids by perioxidation, rendering the cell wall impervious to the triglycerides that are constantly being synthesized within the cell. Thus they accumulate and cause the fatty liver condition previously mentioned. A liver damaged in this manner loses its functional capabilities, such as bilirubin conjugatio-n. This leads to a true classic hepatitis. Serum bilirubin accumulates causing jaundice; hepatic transaminase enzymes lost from the damaged cells cause an elevation in the serum activity. Alkaline phosphatase excretion is impaired and so this enzyme also builds up in the serum. Because they are universally effective, toxins of this type are quickly and easily recognized. Animal exposure is usually under restricted or accidental circumstances. Indirect Hepatotoxins
Whereas the direct hepatotoxins operate in a shotgun-like manner causing a widespread destruction, the indirect hepatotoxins are more selective in their action. Part of this subtlety arises from the fact that immune mechanisms seem to be involved. Consequently, an indirect hepatotoxic effect requires one or more sensitizing doses which are otherwise innocuous. It is the unpredictable second or third or later treatment that is the damaging event, causing post-hepatic cholestasis. The mechanism for this has been circumstantially identified. Histologically, the major bile ducts are dilated by bile plugs and surrounded by eosinophile infiltrations. In the early stages of this disease the parenchymal cells are undamaged and ultrastructural units are intact and *It follows then that the toxicity of such an agent will be dependent on both the amount, or total body burden, of a drug and the rate at which the conversion to a toxin takes place. Since the hepatic drug metabolizing systems are sensitive to chemical stimuli, both stimulating and depressing, the toxic potency of a chemical can vary from animal to animal exclusive of dosage. This important concept will be dealt with in the subsequent article on Drug Interaction.
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will remain unaffected provided the offending chemical is withdrawn. The only lesion found seems to explain the toxic response. Bile duct canaliculi are swollen and distended and apparently obstruct the small bile ducts thus preventing normal bile flow. The drugs most often reported to cause this response are the anabolic steroids, such as methyltestosterone, and long-term administration of phenothiazine tranquilizers, such as chlorpromazine. Literature references that describe indirect toxicity are confined to a small proportion of all exposed patients. Moreover, despite repeated attempts the condition has not been reproduced in animals. This type of hepatotoxicity is unreported in veterinary medicine. Perhaps it is the species restriction; perhaps it is because the population of veterinary patients receiving these drugs is relatively smaller and treatment periods are shorter; or perhaps the condition does occur on a small scale and is unrecognized.
Unclassified This is more than a catch-all category since it includes many of the more recently discovered hepatotoxins that cannot be simply classified as direct or indirect. A case in point is the volatile anesthesic, halothane, that causes a type of hepatotoxicity with features of both the direct and indirect variety. The recognition of halothane hepatitis was troublesome and elusive for many years because of the inconsistent pattern in which it appeared. This drug was u.sed successfully in surgical patients with no suggestion of toxicity appearing until time passed and a considerable number of patients had been reexposed. It was then realized that classic hepatitis with jaundice and serum transaminase elevations occurred in some patients after one or more uneventful exposures. Thus this agent appeared to require sensitization and therefore, it was a typical indirect hepatotoxin. However, the analogy failed when the organ lesion was studied and found to consist of the direct type, zonal hepatocellular necrosis. Moreover, mitochondrial membrane damage and disruption were found by electron microscopy. While all clinical reports of halothane hepatitis have been confined to the human literature, it serves as a classic example of unexpected and incipient drug adversity and provides an objective lesson for practitioners of veterinary mediCine. Another drug-related hepatitis reported to be associated with a specific physiological state is tetracycline-induced, fatty liver of pregnancy. A causal relationship between this condition and tetracycline administration has been difficult to prove and is still disputed. The main point of contention centers around the fact that identical clinical and morphological lesions, jaundice, and fine fatty vacuolization of hepatocytes, have occurred in susceptible women in the last trimester of pregnancy who have not received any medication. In fact, the disease
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was first reported in 1940 before the tetracyclines were discovered. Nevertheless, an increased incidence in patients receiving tetracyclines in late pregnancy has been reported from numerous sources. The use of this class of antibiotics in pregnancy has been for the most part discontinued because of a totally unrelated problem. These agents chelate, or bind, to the calcium in calcified tissues and thus cause yellow tooth discoloration in the offspring of treated mothers. The question of maternal hepatic changes will probably remain unresolved.
NEPHROTOXICITY Prerenal Events Drug-related acute renal failure, classicially characterized by oliguria, is usually the result of a prerenal toxic effect, for example, a hemolytic crisis leading to an excessive load of hemoglobin being presented to the kidneys. A similar condition may also be induced by intramuscular injection of drugs that damage muscle tissue, such as glycerol, wherein myoglobin is released and becomes the destructive agent. Another drug-induced indirect systemic effect is urate nephropathy following anti-cancer drug therapy. The destruction of massive numbers of neoplastic cells releases nucleic acids that are metabolized through normal catabolic pathways to uric acid and its salts. These products have a limited solubility in body fluids which is exceeded and a precipitation of solid crystals within the renal tubules follows. Plugging of the lumina and mechanical damage to the lining cells are the result. A notable example of a countermeasure to a toxic effect has evolved around this problem for it has become a routine practice in human cancer chemotherapy to co-administer allopurinol, a xanthine oxidase inhibitor, which blocks the conversion of nucleic acids to uric acid and thus protects the kidney.
Effects on Glomerulus The glomerulus is rarely subjected to toxic damage despite a high degree of exposure to unbound and therefore, highly active, low molecular weight drug molecules that cross the glomerular membranes. This is fortunate, for the glomerulus, unlike the tubules, is not known to be regenerative. This protection probably arises from the general inert state of glomerular substructures that appear to lack the sophisticated enzyme systems found elsewhere in this organ. Furthermore, both foreign and endogenous molecules pass rapidly from plasma to filtrate so the time of exposure is short. This structure is not completely spared, however. At least one drug has been implicated in causing a specific glomerular toxiCity at prescribed clinical dosages. Dpenicillamine has received scattered reports as a cause of a glomerular nephrosis.
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Effects on Tubules
Down in the lower renal structures, the tubules, where complicated biochemical processes cause drug and water molecules to ebb and flow, the list of toxic substances begins to grow. In the tubules molecular size seems to have a bearing on the structure affected. Heavy metals, such as lead, mercury, gold, and cadmium, damage both the proximal and distal tubules. On the other hand, organic molecules damage only the proximal tubules, and so a list of drugs headed by the antibiotics polymyxin, neomycin, bacitracin, kanamycin, and the tetracyclines are known to cause lesions in these structures, such as cellular necrosis and sloughing, swollen tubules, and intraluminal protein leakage. The type of tubular damage is reflected by functional changes. Resorption of compounds such as glucose, amino acids, and phosphates takes place in the proximal tubules: A typical response to lesions in these structures is glycosuria, aminoaciduria, hyperphosphaturia, and hypophosphatemia. The distal tubutes serve the function of ion exchange and buffering effects, so that damage to these structures is reflected by hyperkaluria and renal acidosis. A more unusual biochemical type of distal tubular lesion has appeared with the advent of lithium treatment in psychiatry. Patients receiving doses causing high serum levels of this element developed a classic diabetes insipidus with polydipsia, polyuria, and a loss of urinary concentrating ability that was unresponsive to injections of antidiuretic hormone. The exact mechanism for this effect is unclear but since lithium is chemically similar to sodium, it suggests the possibility of a competitive antagonism between these two elements for excretory or resorptive mechanisms. Other Effects
At least one compound, methoxyflurane, has the potential for either organic or inorganic toxic effects and this fact has surfaced in clinical reports. The parent molecule of this anesthetic agent can be metabolized in vivo to oxalate, or fluoride, or both; both are nephrotoxins with different mechanisms of action. Excess oxalate causes crystalluria in the proximal tubules. Fluoride nephrotoxicity is more of a biochemical lesion characterized by glycosuria and polyuria without visible lesions. The intrarenal concentrations of the metabolite that accumulate depends upon the relative rate and activity level of the respective enzyme system. Having previously identified the tetracyclines as nephrotoxic agents several additional points should also be mentioned. First is that these drugs deteriorate slowly during storage to form two potent nephrotoxic by-products, anhydro-4-tetracycline and epianhydrotetracycline. Several fatal clinical reactions reported were traced to this effect, permanent reminders of the wisdom in discarding outdated drugs. A second effect tetracyclines have is to cause prerenal uremia, which can be
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serious if it is superimposed on preexisting uremia. This relates to the effect of tetracyclines on blocking protein synthesis. Presumably this prevents amino acids from entering the normal synthetic pathways and instead they are metabolized into nitrogenous compounds such as urea. The liver and kidneys are key excretory organs for removing foreign and toxic chemicals, with a measure of protective interdependence. The results of upsetting this balance are well illustrated by an outbreak of nephrotoxicity caused by the radicicontrast media bunaminodyl and iopanoic acid. These agents are removed by the biliary system and, for this reason, they were developed specifically to outline the gallbladder and bile ducts. As long as the biliary tree was patent no toxic reactions occurred. However, cholestasis caused the m~or burden of drug excretion to fall upon the kidney and cause severe tubular damage in this type of patient. This toxicity mechanism was belatedly demonstrated in dogs with ligated bile ducts. While overt cellular destruction and/or biochemical disruption of critical enzyme systems are primary mechanisms for chemically induced nephrotoxicity, a third system for chemical interference can be the opposite, that of cellular proliferation. This effect caused by the anticancer drug mitomycin C was discovered in the laboratory in time to prevent a similar occurrence in patients. Mice treated with this chemical at clinically subtoxic doses developed hydronephrosis. The cause was traced to a dose-related epithelial proliferation with papilla formation in the renal pelvis which effectively obstructed urine flow to the ureter. The resulting back pressure caused a dilatation and degeneration of the collecting tubules.
HEMATOPOIETIC TOXICITY The toxic effects in this category are manifested clinically as reductions in the level of circulating blood cells, usually erythrocytes, neutrophils or platelets, and occasionally lymphocytes. These changes come about as a result of either a direct action on the mature cell that has entered the blood stream or a depressing effect on the organs that generate replacement cells, the bone marrow, or lymphoid tissues. The type of toxic mechanism involved can lead to differences in the clinical response which are diagnostically important. First is the time lapse following the toxic insult before recognizable changes take place. A direct cellular toxin will destroy the cell quickly, circulating levels will fall rapidly, the number of immature replacement cells will increase and hyperbilirubinemia will occur. When bone marrow toxicity is involved and cell production is halted then the circulating levels will not diminish until normal attrition causes a significant reduction in the circulation, without the appearance of immature replacement cells or elevated bilirubin levels. The short life of a neutrophil, being four or
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five days under normal conditions, means that a drug toxicity which shuts off myelogenesis will not become evident for 48 hours, or when circulating values fall to half the initial levels. A similar erythrocytic effect would not be revealed for a longer period of time since the halflife of this cell is approximately 100 days. With this as a background we shall consider specific examples. Hemolytic Anemias
Anemias can be caused by an agent attacking either the erythrocytic cell membrane or the biochemical interior. Lead and copper are good examples of membrane toxins, since like most bivalent heavy metals, these elements accumulate at the cell surface.* The affected cell loses potassium and at the same time becomes more fragile, lysing in osmotic solutions that would have no effect on healthy cells. Cellular components are also attacked by these agents but presumably in somewhat different ways since copper toxicity is indicated by Heinz body formation, whereas lead causes a basophilic stippling especially in reticulocytes. An organic compound that acts in a manner similar to copper is diphenylhydrazine, a discarded hypotensive drug only rarely used and then to clinically treat polycythemia vera. These direct-acting hemolytic agents appear to damage or alter the red blood cells so that they are prematurely removed from the circulation by the spleen and destroyed. The mature erythrocyte is a metabolically active cell with a limited supply of enzymes. One of these, glucose-6-phosphate dehydrogenase (G-6-PD), is subject to a chemical inhibition that becomes a toxic reaction under predisposing circumstances. All of the cases so far reported have involved humans from specific ethnic backgrounds, either blacks or individuals whose ancestry can be traced to countries bordering the Mediterranean Sea. The red blood cells of these patients have a limited amount of G-6-PD. This enzyme is part of an important biochemical system that reduces or detoxifies the small amount of methemoglobin normally formed in the red blood cell as hemoglobin is oxidized. Several drugs, such as the antimalarial primiquine and the antibacterial nitrofurans and one natural food, fava beans, are active inhibitors of G-6-PD. The combination of minimal levels of enzyme and treatment by an active inhibitor causes the older erythrocytes with higher levels of methemoglobin to be destroyed, presumably by the spleen, leading to a drug-induced hemolytic anemia. This toxicity has been confined to congenitally predisposed humans; therefore, the condition is unknown in other ethnic groups, and seems to be far from the realm of veterinary medicine. It does, however, serve to illustrate a toxicity caused by a drug-gene interaction. *That they accumulate at the cell surface must be remembered when blood samples are taken for analysis of heavy metals. Whole blood is required, never serum or plasma.
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Immune Reactions
Immune mechanisms are important in drug-induced anemias, including the hemolytic variety. One of the most common involving the erythrocyte is the so-called hapten or penicillin reaction. In this example the drug acts as hapten following conjugation with a body protein to form an antigen. In susceptible patients, this stimulates the formation of an IgG type antibody that coats the red blood cell.* Subsequent treatment with the antigenic drug causes an antigenantibody reaction that destroys the erythrocytes. In a second, so-called "innocent bystander" autoimmune reaction, certain drugs, such as phenacetin, chlorpromazine, and some of the sulfonamides, have been reported to induce the formation of a humoral antibody. Additional drug treatments provide a source of antigen and the antibody-antigen combination becomes loosely attached to the red cell surface activating complement and causing cell lysis. Because the attachment to the erythrocyte is tenuous, the antibody is released when the cell is destroyed and becomes free to react with other antigens. This means that relatively small, amounts of antibody can be recycled to cause a disproportionately greater degree of destruction. The third hemolytic mechanism has been labeled the "methyl dopa" reaction after the only drug reported to be involved. Since these patients are Coombs' positive, the most accepted theory explaining this reaction is that the drug somehow alters the erythrocyte rendering it antigenic and causing the production of autoantibodies. Only the immune system has been identified in leading to an active depletion of circulating leucocytes and thrombocytes in clinical cases involving humans. The more diverse and sophisticated mechanisms that destroy erythrocytes are as yet undiscovered. The main body of evidence supporting the hypothesis for leukoagglutinins has come from studies where cells and serum from patients and unaffected humans have been intermixed and the results followed. Usually the combination of normal cells and sensitized serum will produce no response until the offending drug is added, and then a visible agglutination occurs. The further addition of complement causes lysis. Another line of evidence evolves from the lymphocyte transformation test, which, incidentally, shows promise as a diagnostic procedure. In this system, lymphocytes from the patient are mixed with neutral serum containing the drug in question. A typical morphological change in the lymphocyte indicates an immune sensitivity. The drugs most frequently implicated in drug-induced agranulocytosis or thrombocytopenia have been amidopyrine, phenylbutazone, and chlorpromazine. *This is different from the classic penicillin hypersensitivity reaction that involves a serum IgM antibody.
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Effects on Bone Marrow
Agranulocytosis can also be the result of a specific block in myeloid maturation in the bone marrow. This has followed long-term treatment of human patients with high doses of chlorpromazine. In these cases, marrow smears were low in myeloblastic precursor cells. Stem cells were spared and normal function was confirmed by a restoration of neutrophil counts in two weeks following withdrawal of the offending drug. Both myeloid and erythroid cellulopoiesis can be destroyed by drugs, either temporarily or permanently. Three major examples of this toxic panleukopenia are cytotoxic drugs, benzene, and chloramphenicol. Cytotoxic drugs, including the antimetabolites, alkalating agents, and mitotic inhibitors, are primarily the anticancer agents and thus have been selected for one principle activity, potent cytotoxicity. Since they are designed to attack and destroy rapidly dividing cells, it is not surprising that the spectrum of activity includes the bone marrow. This is a well known effect that is detected early. Under most clinical conditions, drug treatments are administered cautiously and halted before the marrow is totally destroyed. At this stage recovery usually follows drug withdrawal. The most serious problem associated with this condition is the susceptibility of treated patients to infections. Therefore antibiotics should be instituted immediately at the earliest indication. Benzene exposure, either by ingestion, absorption of the liquid material through the skin, or inhaling the vapor, has developed a historically notorious reputation for causing complete marrow failure. The destruction included the stem cells and was inevitably fatal to the patient. Because of this. hazard the use of this compound has been severely restricted. This effect was clearly direct since it caused a l 00 per cent incidence following exposure to toxic levels and all species tested showed this response. The mechanism for chloramphenicol-induced aplastic anemia is more obscure. The overall reported incidence of bone marrow toxicity was extremely low with one reaction out of 100,000 patients and only in those individuals receiving high doses for long periods of time. However, the effect was permanent and irreversible so that mortality was high. The only change in blood chemistry that seemed to forewarn an impending toxic crisis was a rise in serum iron with iron binding capacity approaching saturation. Within the marrow, vacuolization of the normoblasts has been reported to be present in affected patients. A few studies have been done with the limited amount of clinical material that could be obtained. An inhibition of protein synthesis by the immature precursor cells has been reported as the primary lesion with a block in the generation of messenger RNA. In recent years, this antibiotic has been used very selectively and the incidence of aplastic anemia has been concomitantly reduced, so the exact nature of this disease will probably never be known.
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Functional Anemias Thus far this discussion of hematopoietic toxicity has considered conditions characterized by a reduction in the number of circulating cells. Another general category is the situation whereby the number of circulating cells is normal but they have been rendered functionally defective by a chemical. The most familiar illustration is methemoglobinemia, or the state of chemically oxidized ferrous ions within the hemoglobin molecule incapable of oxygen transport. At least one drug, the analgesic phenacetin, has been responsible for this toxicity. Another has been environmental nitrates in water supplies or from preserved meats. These are reduced to nitrites which are the toxic agents. The most potent chemicals for this purpose are the aniline dyes. These agents are extremely active so only a minimal exposure is required. For example, there are several reported outbreaks of methemoglobinemia in infants that were traced to the dye used for stamping laundry markings on their. diapers. A type of iron deficiency anemia has appeared in pigs fed cottonseed meal. The active toxin in this diet is gossypol which when extracted and purified causes microcytic, hypochromic anemia and death from pulmonary edema and cardiac failure, but interestingly, only in those species with simple stomachs. This agent has been shown to deplete liver iron and increase bile iron in rats, apparently the result of a chelating property. It has been known for many years that aspirin treatment prolongs clotting time. The exact mechanism for this effect was unknown until recently when it was shown to inhibit platelet aggregation, the first step in clot formation. The details of this experiment are worth considering. The addition of collagen to platelet-rich plasma is followed by a wave of aggregation measurable by appropriate laboratory techniques. The further addition of adenosine triphosphate (ATP) causes another wave of aggregation to occur. It is this second wave that is inhibited by aspirin. Furthermore subsequent studies have shown that this is a functional effect and apparently lasts for the life of the thrombocyte. Platelets transfused from treated donors that previously had received 600 mg of aspirin failed to correct the prolonged dotting time of thrombocytopenic patients until 36 hours after this treatment. At· this point, either the chemical effect was lost or new, unaffected platelets were regenerated and replaced those that were exposed. Spleen Toxicity Finally, in this discussion we shall consider spleen toxicity, a phenomenon reported from laboratory studies. Rats treated with the macromolecular polymers hydroxylamine, polyvinyl alcohol, or methylcellulose developed a normocytic, normochromic, Coombs' negative anemia with reticulocytosis, hyperplastic bone marrow, and spleno-
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megaly. The half-life of untreated red blood cells injected into treated animals was considerably shortened. Furthermore this response was absent in splenectomized rats. Taken collectively, this evidence pointed to a chemically induced, hypersplenism, or a spleen so hyperactive that it destroys erythrocytes long before they would normally expire. The opposite effect, spleen depression or destruction, has been produced in rats receiving single injections of an emulsion of ethyl palmitate, a fatty acid ester. Radioisotope studies have shown that this agent localizes in and destroys the splenic red pulp. One clinical implication of this effect was brought out in studies with rats infected with Hemobartonella muris. Under ordinary circumstances this is alatent infection. The active form, manifested by acute hemolytic anemia, can be precipitated by splenectomy or the chemical under discussion, ethyl palmitate.
GASTROINTESTINAL TOXICITY The gastrointestinal tract is a frequent target for toxins for several very good reasons. First, over and through the mucosal surface of this organ pass the molecules of orally administered drugs in high concentrations. Moreover, if the drug is absorbed and excreted in the bile, then the insult is compounded many times as the agent and/or its metabolites are recycled through the enterohepatic circulation. The second reason for the susceptibility of the gastrointestinal tract is the high rate of cell division constantly underway in this organ. Interference with Mucosal Regeneration
The germinal centers deep in the crypts constantly producing new columnar lining cells are especially vulnerable to cytotoxic drugs, such as the anticancer agents, designed to destroy tissues with rapidly dividing cell populations. For the newly formed cells to move from the crypts to the lining of the villi requires a fixed amount of time. The mature cells that line the villus are in a nondividing, drug-resistant state and they are likely to survive a drug insult and be lost in an uninterrupted pattern. The problem develops when they are not replaced by the drug-suppressed regeneration from the crypt. This simply means that there is a latent period or delayed response between treatment and toxic effect. The action, rather than a direct destruction of the lining mucosa, is one of nonreplacement of the cells which, as part of the normal cell turnover, move up the villar surface and are lost from the villar tip. The clinical effect is a dose-related diarrhea that ranges from loose stools to severe hemorrhagic discharges. As with any diarrhea, fluid and electrolyte losses are serious effects leading to dehydration, shock, renal failure, and death. Replacement therapy is essential and must be maintained until the mucosal lining has regenerated. During this type
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of toxic crisis the patient is also highly susceptible to systemic invasion by entering organisms, so that appropriate prophylactic treatment with antibiotics is in order. Ulcerations
A second m~or type of adverse gastrointestinal effect is drug-induced ulceration of either the upper or lower tract. The anti-inflammatory drugs, such as corticosteroids, aspirin, phenylbutazone, and indomethacin, are the most common causes of drug-induced ulcer in the upper levels of the tract or the stomach and small intestine. Attempts have been made to define the mechanism for this toxicity. Several facts have emerged but no overall answers. The combination of drug and bile appears to be important since in one study no lesions appeared in phenylbutazone-treated, bile duct ligated rats. Furthermore bile collected from phenylbutazone-treated rats was a potent ulcerogenic agent in normal rats. Other lines of investigation have suggested that the permeability of the so-called "mucosal barrier" is altered to permit hydrogen ions to penetrate and damage the submucosal structures. In contrast to the diffuse nature of the cytotoxic-drug type of damage, these lesions are for the most part focal but deeply erosive. Mortality usually results from peritonitis which follows intestinal perforation. Similar ulcers have appeared in the small bowel of patients receiving enteric-coated potassium chloride tablets as a precaution against diuretic-induced hypokalemia. The same type of lesion was experimentally reproduced in dogs and monkeys treated with the potassium salt alone. The exact reason for this response has never been clearly defined. However, the lesions have been reported to include histological changes such as fresh thrombi and fibrinoid necrosis which suggest that the toxic response is one of hemorrhagic infarctions provoked by the sudden release and absorption of high concentrations of potassium within a short segment of bowel. Colitis
A counter response to mucosal destruction, that of stimulated growth, has been reported in humans as pseudomembranous colitis associated with the use of lincomycin and its analogue clindamycin. The initial sign of toxicity has been profuse watery diarrhea in some cases severe enough to cause a profound hypotension. At laparotomy the colon was found to be flaccid and dilated. Both toxic megacolon and colonic perforations have been described. This condition is usually diagnosed by proctoscopic or colonoscopic examination where characteristic raised yellow-white plaques are recognized. The process is reversible following drug withdrawal. The mechanistic cause of this condition is uncertain but, because it is produced by two antibiotics effective against gram-negative anaerobes, especially the Bacteroides
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species, speculation has centered around a possible drug-bacteria interaction. Superinfections A drug-induced, bacteria-mediated toxicity would not be unprecedented and, in fact, has been given the name, drug-induced superinfection. Essentially, two main types have been recognized. One is an overgrowth of fungal organisms, such as the Candida species, the other an imbalance between enteric gram-positive and gram-negative organisms caused by an antibiotic with a narrow spectrum of activity. Symptoms of the latter, which can be experimentally produced within two or three days in rabbits treated with oral penicillin, are profuse watery diarrhea and gas distention of the large bowel. This condition is irreversible unless treated with antibiotics that have a broad or gramnegative spectrum. Pure cultures of gram-negative orgamsms are recovered from the colon of these rabbits. Malabsorption Finally, there is the condition of drug-induced malabsorptive states that has been reported in human cases or experimental animals. Two widely different drugs have had a remarkably similar clinical effect. The drugs are triparanol, an inhibitor of cholesterol biosynthesis, and neomycin, the gram-negative antibiotic. The clinical state is malabsorption of fats and sugars, similar to idiopathic steatorrhea in man, a similarity consistent at the cellular level where a common finding has been villus atrophy accompanied by an increased mitotic activity in the. cryptic mucosa. One notable difference relates to species sensitivity. A condition similar to that found in man has been produced in triparanoltreated rats. Attempts to reproduce the human condition experimentally with neomycin have been unsuccessful. It is beyond the scope of this discussion to present all the evidence that supports or refutes various hypotheses attempting to explain the mechanisms of action for these drugs. For our purposes, it is sufficient to recognize that another mechanism of gastrointestinal toxicity, that of malabsorption, has been amply demonstrated.
EMBRYO-FETAL TOXICITY The thalidomide tragedy of the 1960's focused worldwide attention on the realization that a drug or other chemical can seriously and permanently damage the developing embryo or·fetus without adversely affecting the mother. The most popular solution put forth in the aftermath of this disaster was one of therapeutic nihilism, of simply not treating during pregnancy. While superficially this might appear attractive and reasonable, in actual practice it becomes an oversimplification. The principal coun-
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terargument is based on a fact shared by both human and veterinary medicine, namely, that the developing embryo is most susceptible to toxic chemicals during early pregnancy when many organ systems are rapidly differentiating from primordial tissues. Most importantly this period occurs long before pregnancy is suspected. At issue is a question of whether or not critical therapeutic drugs should be administered to, or withheld from, a female animal because she might have conceived and might be in a critical stage of pregnancy. The more realistic alternative is to use drugs during pregnancy with intelligence, caution, and an understanding of the underlying problems. Stage-Specific Sensitivity To this end, several established principles can be cited. First, is stage-specific sensitivity, meaning that the period of rapid differentiation is virtually the only time that a developing organ can be adversely affected by chemical intervention. The fully differentiated organ is relatively insensitive to the previously toxic stimulus. The exact timing of these events for most pets and domestic animals is unknown, but in laboratory animals such as rodents it has been established that major structures such as heart, central nervous system, and the earliest limb formation begin to appear early in the post-implantation period, i.e., day eight or nine of a 21 day gestation period. On the other hand, cleft palate closure, one of the last major developmental events to take place, can be inhibited by an appropriate insult later in pregnancy, days 14 or 15 in rodents. There are many good practical examples, including thalidomide, that have amply demonstrated the principle of stage-specific sensitivity. An answer to the question "was the organ system which was malformed differentiating when the mother was treated with the drug in question?" can frequently strengthen or refute a suspicion of teratogenicity. Species Specificity Another principle never to be overlooked is that of species specificity. Many chemical teratogens are notoriously specific for a single species and extrapolation to others can be misleading. An interesting example is taken from studies in mice and rats, two species that appear to be close counterparts. Mice treated with corticosteroids during days 13 and 14 of pregnancy produce offspring with a high incidence of cleft palates. Rats are insensitive to this effect. On the other hand, the chemical meclizine hydrochloride, a popular antiemetic drug, given to pregnant rats during the same stage of pregnancy induces the same congenital defect, cleft palate; not so with mice that are resistant. Activation Studies with meclizine hydrochloride in rats and a second cleft palate inducing drug diphenylhydantoin in mice have illustrated another
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important concept that aids in our understanding both teratology and toxicology, that of activation versus inactivation of a toxin. Meclizine hydrochloride is converted to a potent teratogenic metabolite, norchlorcyclizine, by the liver. Concomitant treatment with a drug that depresses the rate of metabolic transformation reduces the number of malformed offspring. Pretreatment with a drug such as phenobarbital that stimulates this same system causes more of the toxin to be generated and consequently increases the number of pups with cleft palate. Diphenylhydantoin causes the same defect in mice for opposite reasons. Here the parent compound is the offender. Inhibited metabolism increases teratogenicity; stimulation reduces the effect. Pharmacological Effects Frequently a drug may adversely influence the fetus by way of its pharmacological properties. Two conditions must be met for this to happen. First, the molecules must cross the placenta, and most active drugs freely cross this barrier by simple diffusion. Second, the fetal organs must be developed and pharmacologically reactive. A familiar example to most practitioners is the depressing effect of maternally administered anesthetics on fetal respiration. Other inadvertent pharmacological effects to the human fetus have resulted from administration of other drugs such as hormones. Certain progestational steroids, having minor unsuspected androgenic properties, given to pregnant females to prevent abortion caused the female offspring to be born with masculine features. Iodides that were part of cough treatment preparations stimulated euthyroid goiters in the offspring. Hypoglycemic agents administered to the mother stimulated insulin secretion by both the maternal and fetal pancreas, and so several babies have reportedly been born showing hypoglycemic convulsions and might have died except for treatment with glucose infusions. Vasopressor substances such as the catecholamines, vasopressin, and ergot alkaloids have caused profound peripheral vasoconstriction leading to anoxia and gangrenous extremities in the fetus. Not surprisingly, the administration of therapeutic anticoagulants to the mother has caused widespread hemorrhaging in the offspring. Drugs can also cause toxic effects in the fetus that are common in adults. The aminoglycoside antibiotics, streptomycin, kanamycin, and gentamycin are well known ototoxins. These have been reported to cause permanent deafness in the children of mothers who were treated with these agents in late pregnancy. ACKNOWLEDGMENTS
The author thanks Drs. Margaret and David Hayden, Veterinary clinician and Veterinary pathologist, respectively, for critically reviewing this and the following article.
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