Module 5: Liver Pathology - Toxic Hepatotoxicities

Module 5 LIVER PATHOLOGY Prepared for the Australian Animal Pathology Standards Program by John Mackie (Vepalabs, Australia) & Roger Kelly (former Reader in Veterinary Pathology, University of Queensland). These modules are designed primarily for candidates who are preparing for Australian College of Veterinary Scientists membership exams… …but they should be useful for undergraduate veterinary students as well.

Module 5 LIVER PATHOLOGY This is the 5th of 5 modules Module 2 Consequences of liver size Sampling (including normal cytology) General pathological reactions Necrosis Module 1 Background, normal structure, developmental anomalies Functions of the liver Failure of function (including clinical chemistry) Module 3 Inflammation Circulatory disturbances Fibrosis Final common pathway of chronic liver disease Viral infections Module 4 Bacterial infections Parasitic infections Neoplasia Module 5 Acute hepatotoxicities Chronic hepatotoxicities

Module 5 LIVER PATHOLOGY Images from various sources have been used in these presentations, and every effort has been made to acknowledge these when they have come from sources other than our own files. We apologise for any inadvertent omissions or errors in attribution, and would be pleased to make appropriate corrections should these errors be brought to our attention. Some of the images have been extracted from virtual sections archived from the National Registry of Domestic Animals pathology (NRDAP). These virtual sections are held by the AAPSP and can be accessed online by subscribers to the AAPSP by using the NRDAP numbers given on the relevant slides.

Module 5. Toxic liver disease The most important agents of toxic liver damage are selectively hepatotoxic… …in other words, they damage the liver while leaving other tissues relatively unharmed. This suggests that either the liver stands as a protective barrier between the absorbed toxin and the other organs… …or that, in performing its natural functions, the liver for various reasons becomes more vulnerable to toxic damage than other organs. As it turns out, selective hepatotoxicities are nearly all the result of the liver’s attempts to extract foreign chemicals from the blood and chemically modify them, in order to excrete them (or prepare them for urinary excretion). If the liver acted simply as a protective barrier, it would be hard to explain why in so many hepatotoxicities the organs of primary exposure (in particular the gut) are not damaged as well.

Module 5. Close-up; gall bladder attachment; sheep poisoned experimentally with Cestrum parqui. Oedema of gallbladder wall; accentuated acinar pattern and petechial haemorrhage. Acute hepatotoxicities Fatal acute hepatotoxicities are characterised by a short clinical illness followed by sudden death. Because of the short clinical course, there is often insufficient time for icterus (or photosensitisation in herbivores) to become clinically apparent before death. The dominant clinical features are the nervous signs of hepatic encephalopathy, and spontaneous bleeding (see back to Module 1 for the reasons for this). Gross necropsy findings are rather stereotyped and non-specific, and all you can hope for is an indication that acute severe liver damage has occurred. The identity of the agent can never be deduced from the gross findings There will usually be more or less severe serosal ecchymosis; slight excess of yellowish or blood-tinged coagulable abdominal fluid; oedema of the gall bladder wall, and some accentuation of the acinar pattern on capsular and cut surfaces of the liver. The carcass may be tinged with jaundice, but this will be unconvincing in breeds/species with yellow fat.

Module 5. The acinar pattern induced by acute fatal hepatoxicity (Cestrum parqui) in a dairy cow. Zonal necrosis results in periacinar cells being replaced by blood after undergoing necrosis. Surviving periportal hepatocytes are paler than normal, hence the striking contrasting pattern. Acute hepatotoxicities (cont.) The basis of fatal acute hepatotoxicity is sudden extensive hepatocellular necrosis. The pattern of necrosis in most acute hepatoxicities is zonal (see Module 2). This explains the prominent acinar pattern seen in most acute hepatoxicities. Of the various patterns of zonal necrosis in these toxicities, by far the most common is periacinar necrosis. This is hardly surprising, since in earlier modules we described how the activity of microsomal mixed-function oxidases (MFOs) varies across the acinus… …and since an important function of these enzymes is to transform xenobiotics into compounds that can be excreted, it is understandable that, on occasion, the intermediate products of these biotransformations are much more reactive than the parent compound. It follows, then, that under these circumstances, the hepatocytes with the highest MFO activity are more likely to expose themselves to dangerous levels of toxic metabolites (so-called “suicidal biotransformations”).

Module 5. Acute hepatotoxicities (cont.) These suicidal transformations are more likely to occur when non-polar compounds (i.e. insoluble in water; soluble in lipid and lipid solvents) get into the cytoplasm of hepatocytes. A classic example is carbon tetrachloride. It is relatively non-toxic to most tissues in its native state, but the reactive intermediate radicals are lethal to the cells that are able to produce them from the parent compound.

Module 5. Why is it that periportal hepatocytes (which, after all, are the first to exposed to incoming blood) are spared in carbon tetrachloride poisoning? Periportal hepatocytes have insufficient MFOs to produce enough reactive metabolites to do themselves harm. Survive. Peracinar hepatocytes have plenty of MFOs; produce lethal amounts of reactive metabolites. Necrosis.

Module 5. Acute hepatotoxicities (cont.) It is possible to stimulate synthesis of high concentrations of mixed function oxidases (MFOs) within hepatocytes. This is a common phenomenon known as induction and can be caused by exposure to a wide variety of xenobiotics (including natural compounds of plant origin) that have to be metabolised for excretion. Their metabolites may not necessarily be toxic. The MFOs increase in concentration and activity in all hepatocytes, but the increasing gradient in activity from periportal to periacinar zones persists. So a sheep that has been exposed to an unidentified plant compound may have undergone MFO induction, and may die of extensive periacinar to pan-acinar liver necrosis when given a dose of CCl4 that would not trouble a non-induced animal. Such idiosyncratic mortalities were relatively common in the days when CCl4 was used widely as a fasciolicide.

Module 5. Acute hepatotoxicities (cont.) However, many of the acute hepatotoxins are water-soluble, yet they still produce periacinar zonal hepatic necrosis (atractylosides from Xanthium, Cestrum; cyanobacterial octapeptides). If they don’t need metabolism to be made toxic, why, then, are the periacinar hepatocytes so vulnerable to these toxins? We’re not sure, but it seems that these cells can preferentially take up some water-soluble toxins by the same mechanism that enables them to preferentially extract bile salts from the sinusoidal blood. This has been shown to happen in poisoning by cyanobacteria (blue-green algae) whose hepatotoxic octapeptides are water-soluble. Who’d be a periacinar hepatocyte? The gross and histological morphological features of acute toxic periacinar necrosis are essentially the same, regardless of the toxin involved, and despite the fact that the mechanism of the damage may be chemically very different.

Module 5. Acute hepatotoxicities (cont.) The list of naturally-occurring hepatotoxins that have been reported to cause acute periacinar necrosis is a long one and is continually being added to. Some examples are below: The liver histology in these intoxications may vary, but the variation has more to do with the dose of the toxin and its duration of exposure than with the chemical structure of the toxin involved.

Module 5. Periportal Acute hepatotoxicities (cont.) The histomorphological features of the hepatocyte necrosis caused by acute hepatoxicity are entirely non-specific Periportal This is illustrated by the range of morphological changes produced by a single dose of an acute hepatotoxin (Cestrum parqui) Viable hepatocytes The changes range from haemorrhagic/coagulative necrosis in periacinar zones to lytic necrosis in the mid-zone Periacinar Since the surviving hepatocytes are restricted to the periportal zone, it is reasonable to assume that periacinar hepatocytes die quickest, with the midzonal hepatocytes taking longer to die Slower necrosis (lytic) It is also reasonable to assume that the slower cells to die have more time to swell and burst (lytic necrosis) Rapid necrosis (coagulative) Periacinar Cestrum poisoning, bovine. The periacinar zone has undergone coagulative necrosis with replacement haemorrhage while the periportal hepatocytes have survived. The transitional zone has undergone lytic necrosis (see later slide). High-power view of the previous section of Cestrum poisoning. Coagulative & haemorrhagic periacinar necrosis, with lytic necrosis at the transition zone

Module 5. papilla. In this view of the duodenal mucosa of a sheep experimentally intoxicated by Cestrum parqui, a trail of mucosal haemorrhage extends downstream from the bile duct papilla. Acute hepatotoxicities (cont.) Experimental intoxication of sheep with Cestrum parqui, Xanthium strumarium (pungens) and Trema tomentosa (aspera) all resulted in acute periacinar hepatocellular necrosis. These intoxications could not be distinguished from one another on the basis of liver histopathology, nor from acute experimental carbon tetrachloride intoxication (A.A. Seawright; unpublished observations). In poisoning by atractylosides in Cestrum parqui, and possibly in other plants, there may be acute haemorrhagic damage to biliary and gallbladder epithelium. This suggests that either the atractyloside itself, or a metabolite of it, is very irritant and capable of being excreted in the bile and damaging mucosal surfaces as it goes. Since injecting this toxin in its native state damages any tissue, it seems likely that it doesn’t need metabolism to be hepatotoxic, and the reason for its selective hepatotoxicity may depend on selective uptake by periacinar hepatocytes.

Module 5. Midzonal necrosis; donkey: suspected Myoporum sp. poisoning Rapid proliferative response by bile ducts and stem cells to a sufficiently severe acute loss of hepatocytes. In this case to acute mid-zonal necrosis, ngaione poisoning in a donkey Zone of necrosis (Image courtesy Lucy Anthenill) Acute hepatotoxicities(cont.) There are some acute hepatotoxicities in which the zonal necrosis is either periportal or midzonal in distribution. These are much less common than those causing periacinar necrosis, and when these uncommon patterns are recognised histologically, the search for the cause of the intoxication is correspondingly narrowed. Only rarely do midzonal hepatocytes undergo selective necrosis, leaving periacinar and periportal hepatocytes more or less intact. One known cause of this distribution of necrosis is poisoning of ruminants by the plant genus Myoporum. The proliferative response by portal stem cells can be apparent within 12 hours of onset of an acute necrotising hepatocellular episode, irrespective of its zonal distribution. But this response is particularly brisk after periportal or mid-zonal necrosis. (Image courtesy Lucy Anthenill)

In this case and in field cases of M. insulare intoxication, the zonal necrosis was consistently periportal. Variation in zonal distribution may be related to differences between Myoporum species, or to presence or absence of inducing components of the diet. Module 5. Patchy variation in distribution of acute midzonal/periportal necrosis in a calf experimentally intoxicated with Myoporum insulare (boobialla). Patchy distribution of changes in experimental M. Insulare poisoning, calf. Photo courtesy Ian Jerrett. For details, see Jerrett and Chinnock, AVJ 60, pp183-186 (1983). Acute hepatotoxicities(cont.) Among mortalities in a mob of sheep exposed to Myoporum deserti, liver damage may range from periacinar to midzonal, or from midzonal to periportal. The oil responsible is ngaione. In some animals, the pattern of necrosis and its severity vary throughout the liver, imparting a patchy appearance to the surfaces. Alan Seawright found that the distribution of the zonal necrosis in mice caused by ngaione could be changed, either by suppressing the MFOs or by inducing them. The necrosis tended to be periportal in induced mice, periacinar in suppressed animals, and midzonal in controls. This is not a phenomenon you will see often in pathology practice, but it serves to illustrate the importance of the MFO system in hepatoxicity. (Seawright, A.A., and Hrdlicka, J. The effect of prior dosing with phenobarbitone and ß-diethylaminoethyl diphenylpropyl acetate (SKF525A) on the toxicity and liver lesion caused by ngaione in the mouse. British Journal of Experimental Pathology53: 242-252, 1972.)

Periportal necrosis: cow. “Acute bovine liver disease” (ABLD) Module 5. Hepatic venule Hepatic venule Acute hepatotoxicities(cont.) We have seen that the zone of necrosis can be moved from periacinar to periportal in ngaione poisoning in laboratory animals, and that periportal necrosis might be expected as well as midzonal necrosis in some individuals in natural outbreaks of Myoporum poisoning. However, there there a few acute hepatotoxicities in which the necrosis is predictablyperiportal. Allyl alcohol can do it experimentally, and white phosphorus (in baits) could produce a pattern of liver damage that was mostly periportal: these intoxications are unlikely to be seen these days. But there is a significant bovine hepatotoxicity in which the necrosis is predictablyperiportal. At least, we assume it is a hepatotoxicity, although neither the toxin nor its source have been identified. So it is called, for want of a better name, “Acute Bovine Liver Disease” (ABLD). It is seen in beef or dairy cattle of any age, on pasture in spring or autumn under warm, humid conditions. Portal triad Outbreaks have often occurred on pastures infested withsenescent Cynosurus echinatus (rough dog’s tail grass).

Module 5. Probable convalescent case of ABLD with early fibrosis and bile duct proliferation about portal triad. Hepatic venule Portal triad Acute Bovine Liver Disease (ABLD; continued) Sudden onset of photosensitivity, pyrexia, decreased lactation. One or more sudden deaths, +/- hepatic encephalopathy, icterus. Photosensitivity and ill-thrift persist; often necessitating culling. Bile duct proliferation and fibrosis evident in presumed recovered cases. Feeding trials with Cynosurus echinatus have been negative. The toxicity of the involved pasture seems to be transient: cases are generally seen 12-24 h after introduction to spelled pasture.

Module 5. Recovery from acute toxic liver necrosis If a single acute hepatotoxic insult isn’t fatal, complete recovery can occur, with restitution of normal gross and microscopic liver structure and return to clinical good health. For the same severity of zonal necrosis, it seems this repair will be more rapid and complete in cases of periacinar compared to periportal necrosis. This is probably because, in the case of periportal necrosis, it will be more difficult to re-establish connections between canaliculi and cholangioles, and, moreover, persistent portal fibroplasia is likely to be more severe. This might explain why in ABLD the intoxication, although apparently transient, results in persistent cholestasis and ill-thrift.

Module 5. Acute hepatotoxicities (cont.) Acute hepatotoxicity and massive necrosis. Regardless of the zonal pattern of acute necrosis produced by any hepatotoxin, if the dose is high enough, the result will be more or less massive necrosis (see Module 2 for definition). Under these circumstances, the massive necrosis can be regarded as simply the most extreme form of zonal necrosis. If widespread sampling of such a liver is undertaken, there will usually be some areas of zonal necrosis. These should be sought, since they may allow distinction of periacinar from periportal or midzonal necrosis. Massive (panacinar) necrosis; cyanobacterial intoxication; sheep. Other sections showed severe periacinar necrosis.

Module 5. Chronic hepatotoxicity Chemical attributes of chronic hepatotoxins The chemical range of compounds capable of selectively causing chronic liver injury is broad. Some are acutely hepatotoxic but can produce chronic liver damage when sublethal doses are repeated (e.g. carbon tetrachloride). The more important ones, though, can produce injury after only one exposure, and this damage tends to be persistent, and tends to be limited to the liver. What chemical characteristics do these often quite different toxins share?

Module 5. Chemical attributes of chronic hepatotoxins To be chronically hepatotoxic, a compound obviously must first to be taken up by the liver. Most are then transformed to some extent by the microsomal mixed-function oxidase activity of hepatocytes into one or more reactive metabolites (as was described for some acute hepatotoxins). However, for chronic hepatotoxicity to occur after a single dose of such a compound, the reactive metabolite, in addition to being harmful, must also resist inactivation and persist in the hepatocyte, sometimes for weeks or months. It will therefore be able to exert its effect for a considerable period.

Module 5. This table lists some of the more important chronic hepatotoxins in domestic animals.

Module 5. Effects of chronic hepatotoxins Chronic hepatotoxicity will not be clinically apparent until it causes one or more liver functions to fail (see Module 1). The clinical signs of liver failure due to chronic hepatoxicity overlap those of acute hepatoxicity, but are slower in onset, and bleeding due to consumption coagulopathy is less likely to occur. Icterus, when caused by chronic hepatotoxicity, is usually more severe than that seen in acute hepatoxicity because it has more time to develop. However, bile excretion may be relatively unimpaired in some chronic hepatoxicities, so absence of icterus does not rule out severe chronic hepatoxicity.

Module 5. Effects of chronic hepatotoxins Likewise, photosensitivity is more likely to accompany chronic hepatoxicity than it is acute hepatotoxicity (more time for it to develop), but will not be a feature in those cases of chronic toxicity in which phylloerythrin excretion is relatively unimpaired... ...so the absence of photosensitisation does not necessarily rule out chronic hepatotoxicity. On the other hand, severe icterus and photosensitivity do not necessarily indicate that death was due to total liver failure. For example, in Lantana poisoning of ruminants (see later), failure of bile salt and phylloerythrin excretion may occur, yet death is likely due to a combination of gut stasis, dehydration and renal, cardiac and liver damage, and not solely due to failure of vital liver functions... ...as evidenced by the fact that affected animals can be saved by protection from light, careful rehydration and administration of activated charcoal to remove the toxin (which at least indicates the liver damage is reversible).

Module 5. Effects of chronic fatal hepatotoxicities Chronic hepatotoxicity will usually result in an increase in the proportion of stroma to parenchyma, with or without reduction in liver size. Therefore, some restriction of portal blood flow is almost inevitable. So portal hypertension is a very common finding in late stages of chronic hepatoxicity, as evidenced by copious ascites. Portal hypertension in cattle can cause intestinal oedema with consequent malabsorption. So the clinical features of chronic hepatoxicity in cattle can include severe intractable diarrhoea with straining and some rectal prolapse. Large intestinal oedema; bovine. Congestive heart failure and chronic liver disease can both produce this. Some degree of malabsorption is inevitable, so diarrhoea can distract the diagnostician from the underlying cause.

Module 5. Effects of chronic fatal hepatotoxicities The portal hypertension may lead to development of acquired porto-systemic shunting (see Module 3). This in turn will exacerbate hepatic encephalopathy, which may already be manifest in affected animals. Appetite suppression and general metabolic derangement cause severe inanition. By this time, the enormous reserves of most important liver functions have been exhausted, essentially irreversibly. The most effective management option at this stage is diagnosis by necropsy in the hope of detecting the cause and avoiding it in future.

Module 5. The diagnostic challenge of chronic hepatotoxicity: summary We have seen that wasting, ascites and a tougher than normal liver are to expected in cases of fatal chronic hepatotoxicity. The liver itself may be of normal shape and colour, and merely slightly smaller and tougher than normal. Or it may be nodular (see later). Surprisingly, bile excretion may be maintained during the terminal stages, so icterus may be not severe enough to be noted grossly. If wasting, ascites and nervous signs are the dominant clinical signs, then it is not surprising that chronic hepatotoxicity is sometimes mis-diagnosed at field necropsy. Even laboratory blood results may not help much, since serum/plasma levels of the common indicators of liver damage may not be markedly altered in chronic hepatotoxicities. Serum bile acids may be elevated. Other biochemical tests of liver function, such as BSP clearance or blood ammonia levels, are rarely performed because of expense.

Module 5. Chronic hepatotoxicities: pyrrolizidine alkaloid (PA) poisoning PAs vary greatly in their chemical structure and toxicity. They are found in a large number of species of several genera of plants (e.g. Crotalaria, Senecio, Heliotropium, Echium). Different animal species vary greatly in their susceptibility to a given toxic PA. Juveniles are more susceptible than adults. Experimental administration of a very high dose of PA can produce acute periacinar hepatic necrosis, but this is never seen under field conditions. PAs are metabolised by hepatic microsomal enzymes to long-lived reactive intermediate adducts that bind tenaciously to hepatocellular macromolecules. These adducts (bound pyrroles) can exert chronic toxic effects for many weeks after a single dose. Their demonstration in liver and other tissues can be used for confirmation of exposure to the toxin. An important feature of PA poisoning is reduction in the normally high mitotic/regenerative capacity of the liver.

Module 5. Chronic hepatotoxicities: PA poisoning (contd.) In severe intoxications in susceptible individuals, this antimitotic effect prevents normal replacement of hepatocytes at the end of their life cycle. This results in the liver becoming uniformly smaller, paler and tougher (due to condensation of pre-existing stroma +/- de novo fibrosis). In the terminal stages there is usually enough portal hypertension to cause some ascites, especially in cattle. Affected animals succumb to liver failure, usually without much icterus or photosensitivity.

Module 5. Abattoir specimen, ovine. The animal may have been unthrifty, but was clearly fit enough for sale and slaughter. Bovine liver; abattoir specimen. Again, an animal fit enough to make it to slaughter. Chronic hepatotoxicities: PA poisoning (contd.) The antimitotic effect of PAs is not equally expressed in all hepatocytes throughout the liver. Therefore, in later stages of less severe intoxications, under the stimulus of parenchymal loss, some groups of hepatocytes escape the antimitotic effect and begin to proliferate. Because these foci of proliferation are randomly distributed, the result is an attractive mosaic of nodular regeneration. The regenerative nodules may be paler than surrounding parenchyma (due to fatty change), and are sometimes discoloured by bile retention. The implication is that these regenerative nodules are dysfunctional relative to normal parenchyma. Because these foci of proliferation are randomly distributed, the result is an attractive mosaic of nodular regeneration. Generalised micronodular proliferation, with a larger nodule discoloured by bile retention (arrow).

Module 5. Chronic hepatotoxicities: PA poisoning (contd.) If a sheep flock is exposed to PA-containing plants during drought, deaths from liver failure are typically delayed for several months while the liver slowly loses mass and function. Livers from such animals may be smaller and tougher than normal because of loss of parenchyma and condensation of normal stroma. The degree of nodular regeneration is very variable and may not be evident. When present, the regenerative foci can be mistaken for inflammatory or neoplastic foci. Chronic PA poisoning, ovine (abattoir specimen). The flock was culled due to non-specific ill-thrift. The liver appears relatively normal apart from the small regenerative nodules. The degree of parenchymal loss is subtly suggested by the finely-wrinkled and slightly thickened capsule. Section of the liver in the last slide. The hyperplastic foci vary in size, are randomly distributed and are paler than surrounding parenchyma. Image courtesy of Rod Badman Image courtesy of Rod Badman

Module 5. Chronic hepatotoxicities: PA poisoning (contd.) Histopathology Text-book descriptions of liver histology of PA-intoxicated livers include the following: • Megalocytosis, karyomegaly • Bile duct proliferation • Hepatic venular fibrosis • Portal fibrosis • Absence of mitoses • Intranuclear inclusions (cytoplasmic invaginations) • Nodular hyperplasia Unfortunately, all these changes may be seen in livers that have been exposed to other chronic hepatotoxins, so caution is required when trying to diagnose PA poisoning on the basis of histopathology alone. Megalocytosis, for example, was once thought to reliably indicate PA poisoning, but we now know that it is seen in many other chronic hepatotoxicities. Likewise, florid fibrosis of hepatic venules (“veno-occlusive disease”) was first described in Crotalaria poisoning in human infants, and was thought to be specific for PA poisoning. But it has also been seen in bovine cycad poisoning (see later).

Module 5. Chronic hepatotoxicities: PA poisoning (contd.) Histopathology The histomorphological outcome of PA poisoning in any individual is influenced by a daunting list of factors, including: This variability was beautifully illustrated in a line of weaner beef cattle from northern NSW that were culled for slaughter because of ill-thrift after several died due to poisoning by Senecio lautus (fireweed). The livers were sampled at slaughter for histopathology. The variation in liver histology in this cohort of animals was startling and is depicted in following slides. (blocks kindly provided by Roger Cook)

Module 5. Chronic hepatotoxicities: PA poisoning (contd.) Histopathology (remember; the following images were taken from samples from the same outbreak) (same outbreak as previous slide; different animal) There is less karyomegaly and megalocytosis in this liver, but there is quite severe periacinar and portal fibrosis. Hepatic arterioles are numerous and prominent in the triad, which suggests that the fibrosis has caused significant portal hypertension with acquired porto-systemic shunting. The most obvious changes in this liver are the variable enlargement of hepatocellular nuclei and cytoplasm, with coarsening of nuclear chromatin and nucleolar enlargement (karyomegaly and megalocytosis). Note the slight basophilia of perinuclear cytoplasm in conjunction with these changes: this has been held to be pathognomonic of PA poisoning (a plausible but unproven hypothesis).

Module 5. (same outbreak as previous slide; different animal). In this liver there is little karyomegaly or megalocytosis and changes in portal triads are minimal, but hepatic venules are sometimes obliterated by loose, florid fibrosis like that seen on the left. This fibrosis may be caused by toxic metabolites spilling into sinusoids and venules from hepatocytes and damaging downstream endothelium. This is unproven in cattle, but endothelial destruction has been shown experimentally in chickens (see following slides). (same outbreak as previous slide; different animal) In this liver, there is obvious karyomegaly and megalocytosis as well as portal fibrosis but no periacinar (perivenular) fibrosis. There is mild bile duct hyperplasia.

Module 5. TEM, normal perfusion-fixed chicken liver, with delicate endothelial extensions in orderly and intimate apposition to hepatocellular microvilli. (courtesy M. Ghoddusi) Kupffer cell Endothelial cell cytoplasmic extensions

Module 5. TEM, perfusion-fixed chicken liver, acute PA poisoning (monocrotaline;12hr) (courtesy M. Ghoddusi) Hepatocytes still viable, but have lost their microvilli Only tattered remnants of endothelial and Kupffer cells

Module 5. Chronic hepatotoxicities: phomopsin poisoning (lupinosis) Gross pathology The gross changes in phomopsin poisoning of sheep and cattle are non-specific (see the introduction to chronic hepatotoxicity above). Liver pallor, atrophy and nodular hyperplasia may occur, so the gross pathology can be very similar to that of PA poisoning. Icterus and photosensitivity may be more consistently seen than in PA poisoning, but it is not possible to distinguish PA from phomopsin intoxication grossly. Both lupinosis and PA poisoning will predispose sheep to the haemolytic crisis of chronic copper poisoning (for reasons, see copper poisoning, below), in which case icterus will be predominantly haemolytic and severe.

Module 5. Phomopsin poisoning, sheep Abnormal mitotic figures Chronic hepatotoxicities: phomopsin poisoning (lupinosis) Histopathology The histological changes in phomopsin poisoning of sheep and cattle are similar in many respects to those of PA poisoning. They include karyomegaly, megalocytosis, fibrosis, nodular hyperplasia and biliary hyperplasia. A distinguishing feature of phomopsin poisoning is the presence of abnormal mitotic figures in hepatocytes. These appear to be mitoses that have been arrested in metaphase, and their presence is of diagnostic significance. Specific features include abnormally dispersed or clumped chromatin. Additional features may include variably severe hepatocellular fatty change and increased single cell necrosis. Periacinar necrosis and pigmented debris

Module 5. Chronic hepatotoxicities: chronic copper poisoning (sheep) Sheep (especially British breeds) are adapted to environments with low Cu availability, so tend to store excess Cu in liver when it is easily available. Their hepatocytes have remarkable avidity for the element. Excess uptake can be due to an excess of Cu in the diet, or to deficiency of dietary molybdenum Cu is stored in hepatocyte lysosomes where it has little effect in concentrations of up to about 1,000 ppm (dry weight). At higher concentrations, it causes individual hepatocytes die with increasing frequency. This single cell necrosis is marked by small accumulations of neutrophils. Further increase in liver Cu is associated with generalised hepatocyte swelling, nuclear enlargement with nucleolar prominence, and accumulation of pigmented macrophages in sinusoids and portal stroma. Affected animals may be clinically healthy, even as liver Cu concentration rises through 3,000 ppm. But liver-specific enzymes and Cu levels in serum will be rising... Focal necrosis in the asymptomatic (accumulation phase) of chronic Cu poisoning in a sheep. Hepatocyte death seems either to be by apoptosis (white arrow) or is attended by small but intense inflammatory cell accumulations (yellow arrows). (NRDAP 0198; ovine) ...because hepatocytes will be dying with exponentially-increasing frequency. At this stage, however, hepatocyte mitosis is keeping pace with the death-rate and the youngsters are sweeping Cu out of sinusoidal blood (they can’t help themselves). Pigmented macrophages have accumulated, mostly in portal stroma (arrows). There is karyomegaly and megalocytosis, so PA exposure could have been involved, although some megalocytosis will occur in CCP without PA involvement. Hepatocyte degeneration is most severe in the periacinar zone (lower right), suggesting that haemolysis may already have begun with subsequent anaemia. (NRDAP B0168; ovine)

Module 5. Chronic hepatotoxicities: chronic copper poisoning in sheep (contd.) When the hepatocyte death rate exceeds the replacement rate, copper is released into the sinusoidal blood at an exponentially increasing rate. Plasma Cu levels eventually rise high enough to cause intravascular haemolysis, which rapidly increases in severity. The hypoxaemia caused by massive haemolysis results, unsurprisingly, in progressive periacinar necrosis, with further dumping of Cu into the circulation. So a vicious cycle is initiated. So within a few hours, a previously “healthy” sheep can be dying of a combination of paroxysmal haemolytic anaemia and liver failure. Clinically, there is sudden severe depression, haemoglobinuria and jaundice (too acute for photosensitisation to occur). Necropsy findings are dominated by severe jaundice (hepatocellular and haemolytic), dark flocculent bile, enlarged dark spleen, “gun-metal” kidneys, red urine and a pale liver stained orange by bile and free haemoglobin.

Module 5. Chronic hepatotoxicities: chronic copper poisoning (sheep, cont.) We have seen how survival of copper-loaded sheep is dependent on the capacity of the dying hepatocytes to be replaced by mitosis. It follows that anything that interferes even slightly with effective hepatocyte replacement will bring forward the onset of the haemolytic crisis Pyrrolizidine alkaloids are often associated with outbreaks of the crisis of Cu poisoning because of their potent anti-mitotic activity. The same is true of phomopsin. But slight stresses such as brief feed deprivation during mustering can be enough to trigger the crisis in highly loaded individuals. Highly copper-loaded sheep are haemolytic crises waiting to happen. Many accounts of CCP in sheep imply that PA-poisoned livers have greater affinity for Cu than normal livers. Good evidence for this is lacking. As for diagnosis, remember that 3500 ppm Cu in a sheep’s liver may not have killed it. It may have been killed by lightning during the preclinical phase. So determination of renal Cu content is best for confirmation, because it rises markedly during the haemolytic crisis.

Module 5. Image courtesy Rachel Burns Chronic hepatotoxicities: chronic copper poisoning in dogs Some dogs are genetically predisposed, like sheep, to accumulate hepatotoxic burdens of copper in their livers. The heritable nature of the canine condition was first established in Bedlington terriers, but affected individuals are occurring in an increasing breed range. Although there may be a haemolytic conclusion to the disease in dogs, this is unusual. Typically, the disease is characterised by progressive hepatocellular degeneration, fibrosis and nodular remodelling progressing to chronic liver failure with portal hypertension and ascites. Histologically, the degeneration is usually accompanied by chronic inflammatory infiltrates, sufficient to divert suspicion toward infectious agents. Regenerative nodule of younger hepatocytes Inflammatory infiltrates There is little to distinguish the histopathology from that of the confusing array of chronic canine hepatitis entities that were discussed in Modules 2 and 3, except for the presence of eosinophilic cytoplasmic granules that contain copper, demonstrable by special stains such as rhodanine.

Module 5. Chronic hepatotoxicities: indospicine poisoning in dogs This is another chronic canine hepatoxicity in which there is a significant inflammatory component. Indospicine, a toxic amino acid in various Indigofera species, accumulates in meat and tissues of horses, camels and probably other species that graze these plants. Dogs eating this meat also accumulate the toxin and some (not all) develop chronic liver damage. Livers of most other species are unaffected by this amino acid unless at (experimentally) very high dose-rates. Horses, however, develop nervous signs, probably caused by another component of the plant (3-nitropropionic acid). But they don’t develop significant liver disease. The liver changes in dogs are characterised histologically by progressive periacinar degeneration and necrosis which is accompanied by a grumbling mononuclear inflammatory infiltrate. This may progress to parenchymal collapse, cholestasis and some fibrosis, leading eventually to liver failure. Chronic indospicine poisoning, dog Progressive single-cell necrosis and mononuclear infiltration in periacinar zone.

Module 5. Chronic hepatotoxicities: sporidesmin intoxication Ruminants and camelids (and possibly kangaroos) are susceptible; typically on senescent ryegrass pastures infected by Pithomyces chartarum in warm humid weather. This fungus produces the mycotoxin sporidesmin. Sporidesmin can produce acute periacinar hepatocellular necrosis, but only experimentally at high doses. Sporidesmin is directly toxic if applied to any tissue and does not require metabolism for toxicity. Its hepatotoxicity derives from the fact that it is concentrated in bile. The irritant effect is thus concentrated in portal triads, with severe bile-duct damage and proliferation; periductular oedema and fibrosis dominating the histological changes. There may also be portal vascultis and periportal hepatocyte degeneration. The bile-duct damage results in severe obstructive jaundice with consequent hepatogenous photosensitisation (“facial eczema”; see Module 1). In camelids, sporodesmin causes bile duct proliferation and portal fibrosis on an even more majestic scale than it does in ruminants. Florid bile ductular proliferation and portal fibrosis; ovine facial eczema (NRDAP B0367)

Module 5. CHRONIC SPORIDESMIN TOXICITY (“FACIAL ECZEMA”), BOVINE Enormously hypertrophied right lobe, with capsular scarring extending from portal fibrosis Chronic hepatotoxicities: sporidesmin intoxication As with any chronic generalised long-term interference with bile drainage in ruminants, the left liver lobe is very likely to undergo atrophy and fibrosis. The remaining liver will attempt to replace the lost parenchyma, and the end result will be an extremely misshapen organ. Outbreaks of sporidesmin poisoning can be very similar clinically to steroidal sapogenin poisoning (see next slide). However, since bile ducts are not the primary target of sapogenins, distortion of the liver is not produced in chronic cases of sapogenin intoxication. Atrophic leaf-like left lobe, folded back on itself

Module 5. Chronic hepatotoxicities: steroidal sapogenin poisoning (formerly known as crystal-associated cholangiohepatopathy) Bile ductular proliferation Naturally-occurring intoxication has been reported in sheep, cattle & goats. Crystal clefts Steroidal sapogenins are produced by some plants most of the time, and by many other species unpredictably. Some useful pasture species (Panicum, Brachiaria spp., Tribulus terrestris) can become temporarily toxic under certain poorly-understood circumstances, due to their unpredictable production of steroidal sapogenins. Cholestasis and hepatogenous photosensitivity are the principle clinical features, with minimal interference with other liver functions. So while photosensitivity may be severe, total liver failure does not occur. Histological changes in the liver are quite subtle, consisting mostly of bile ductular (stem cell) proliferation. There may also be Kupffer cell hypertrophy and hyperplasia and mild cholangitis. The only specific histological finding is the presence of needle-shaped to lenticular clefts in hepatocytes, Kupffer cells and bile ducts caused by sapogenin crystal deposition. Steroidal sapogenin poisoning, goat (AAPSP PT December 2008 - Case A)

Module 5. Chronic hepatotoxicities: steroidal sapogenin poisoning These crystals are mostly removed during tissue processing, but optically active fragments may persist in heavy deposits. It was once thought that the cholestasis was due to mechanical obstruction of bile ducts by the crystals. It is important to recognise, however, that crystals or their spaces may not be present in some cases of sapogenin-induced photosensitivity. In Brachiaria-derived sapogenin poisoning in cattle, the characteristic histological feature is foamy macrophage accumulation, rather than crystal formation. Grossly, affected livers of sheep and cattle may be bile-stained but are not distorted or fibrotic (in contrast to sporidesmin-poisoned livers). Bovine liver, Brachiaria sp. poisoning. Two focal aggregations of foamy macrophages in parenchyma. It’s tempting to attribute cholestasis to mechanical blockage by crystals, but the crystals are not necessary for cholestasis.

Module 5. Bile ductular proliferation (seen in more chronic cases) Chronic hepatotoxicities: Lantana poisoning (cattle & sheep) Lantana poisoning is similar to sapogenin intoxication in that it is primarily a cholestatic condition, with other liver functions more or less preserved. Affected animals can conjugate bile but there appears to be paralysis of canalicular transport (in EMs, normal canaliculi are often hard to find). The liver changes can be surprisingly subtle, even in profoundly icteric and photosensitised cases. Histologically, they are characterised by hepatocellular enlargement, vacuolation and minimal stem cell proliferation. Periacinar canalicular cholestasis may be present. The triterpenes responsible also damage renal tubules and cause profound gut stasis. The gall bladder is also paralysed and becomes distended with pale mucoid bile Kidney damage takes the form of acute tubular nephrosis, which with the gut stasis contributes to life-threatening dehydration. Marked enlargement of hepatocytes, with granular cytoplasm, vacuolated periportally. Canalicular cholestasis not present in this case, but may be present in periacinar zone. Lantana poisoning; bovine liver. Pallor and bile-staining of capsular and cut surfaces, that’s all. Nephrosis in bovine lantana poisoning (NRDAP A0096) Bovine lantana poisoning: NRDAP B0109

Module 5. Other chronic hepatotoxicities: Aflatoxin, MAM, nitrosamines, etc Bovine Lepidozamiaperoffskyana poisoning.Periacinar fibrosis These intoxications produce the range of gross liver changes expected in chronic liver disease of any cause... ... fibrosis, pallor, nodular regeneration and atrophy of the organ. The microscopic changes are likewise nonspecific, so the cause cannot with confidence be based on histopathology. The next slide is of a chemically-confirmed case of bovine aflatoxicosis that a panel of experienced pathologists thought was pyrrolizidine alkaloid poisoning. It shows obvious karyomegaly. Likewise, hepatic venular fibrosis (“veno-occlusive disease”) and many of the other changes seen in PA poisoning can be produced by methylazoxymethanol from cycads. The moral of this story is to recognise that the final common pathway of most chronic hepatotoxicities tends to obscure any histologically pathognomonic features. So analyse for chemical residues if you can: they (or their foot-prints) will probably be there. (Image courtesy R. Cook)

Module 5: Liver Pathology - Toxic Hepatotoxicities