1 Cellular injury
All mammalian cells strive to survive against a hostile fluctuating environment by expending energy to maintain a tightly regulated internal and local external environment. If the environmental fluctuations are sufficiently large, they will change the state of the cell, which will then attempt to return to its usual condition. Cellular injury, manifest as a significant disturb-ance of cell function and central to almost all human disease, occurs if the changes in the cell are sufficiently large. In any particular case it may be difficult to tell whether a measured change is due to damage or is due to some meaningful response on the part of the cell.
On the whole, (b) is the least likely because cells are capable of significant reparative processes, and if they survive an insult, they generally repair it; if the damage is not lethal but is very severe or persistent and beyond the capacity of the cell to regenerate, the cell may activate mechanisms that result in its own death.
Certain injurious agents (radiation, certain chemicals, viruses, and some bacterial and fungal toxins) directly damage the cell nucleus and deoxyribonucleic acid (DNA), resulting in genetic DNA mutations. Depending on the degree of damage and the portion of the DNA damaged, the damage may be reparable, resulting in a temporary cell cycle arrest but ultimately no phenotypic alteration. Severe irreparable damage triggers apoptotic pathways that culminate in cell death. An intermediate degree of DNA damage results in genetic mutations that do not directly impair cell survival and may confer a survival advantage. Successive mutations will then drive the cell down the multi-step pathway towards neoplasia. The processes involved in oncogenesis are described in Chapter 5.
The cell is a highly-structured complex of molecules and organelles that are arranged to fulfil routine metabolic housekeeping functions and the specialised functions that make one cell different from another. In order to carry out these functions the cell has energy needs and some transport mechanisms to facilitate the import of metabolites and the export of waste products. Injury to a cell results in relative disruption to one or more of these structures or functions.
The microscopic appearance of damaged cells is sometimes characteristic of a particular cell type but is seldom specific to the type of damage. When we refer to changes in appearance, we are talking about the appearances seen on histological preparations stained with various dyes; this is, of course, a long way from the biological processes that have caused the cell changes. It must also be remembered that many of the features seen in routine histological preparations are the result of artifacts induced by fixation, tissue processing, and staining and may not directly represent the appearance of the cells in vivo. We must also consider that when a tissue is injured, morphological changes take time to develop. For example, if a patient suffers the sudden occlusion of a coronary artery due to a thrombus, the cardiac myocytes will die within just a few minutes. However, if the patient suffers a fatal cardiac arrhythmia within the first hour of the infarct, no morphological features may be present to indicate that myocyte damage has occurred, either macroscopically or histologically. Nonetheless, a consideration of such changes is valuable when compared to the histology of the normal, uninjured, cell.
Cellular damage that affects the membrane-bound ion pumps results in a loss of control of the normal cellular ionic milieu. The unregulated diffusion of ions into the cells is accompanied by a passive osmotic influx of water. Consequently the cell swells as the cytoplasm becomes diluted. Histologically these damaged cells have a pale swollen appearance in haematoxylin and eosin-stained sections.
This is a characteristic change seen in liver cells as a response to cellular injury from a variety of causes. Under the microscope the cells contain many small vacuoles finely dispersed through the cytoplasm, or a single large vacuole that displaces the nucleus. These are known as microvesicular and macrovesicular steatosis, respectively. The vacuoles are empty because in life they contained fat which dissolves out of the sections during histological processing, leaving a hole. It is possible to identify the substance in such vacuoles by cutting sections from fresh frozen tissue. This does not involve exposure to fat solvents; the contents of the vacuoles can then be demonstrated using specific fat stains such as Sudan black or Oil red O. Fatty change in the liver occurs as a result of damage to energygenerating mechanisms and to protein synthesis since fat is transported out of the cell by energy-dependent protein carrier mechanisms and damage to these results in passive fat accumulation. The most common cause is exposure of the hepatocytes to alcohol.
Haematoxylin stains acids such as deoxyribonucleic acid (DNA) and ribonucleic-acid (RNA), and eosin stains proteins (proteins are amphoteric but contain many reactive bases). The cytoplasm contains proteins and RNA among other things. Cellular damage often results in a diminution of cytoplasmic RNA, and thus the colour of such cells becomes slightly less purple and more pink (eosinophilic). This is a characteristic of cardiac myocytes in the early stages of ischaemia and may often be the only histologically visible change in postmortem tissue. Eosinophilic change must be distinguished from oncocytosis, which also causes cells to have a profoundly eosinophilic and finely granular cytoplasm due to the accumulation of mitochondria within the cytoplasm. Oncocytic change is seen on occasion as a metaplastic process within the endometrium, but a number of neoplasms including those in the kidney, have oncocytic variants.
These may be subtle, such as the disposition of chromatin around the periphery of the nucleus, often referred to as clumping, or more extreme alterations such as condensation of the nucleus (pyknosis), fragmentation (karyorhexis) and dilatation of the perinuclear cisternae of the endoplasmic reticulum (karyolysis). A small circular structure, the nucleolus, becomes more apparent as the nucleus is activated; this is the centre for the production of mRNA. The nucleolus can be demonstrated by silver stains (the resulting granules being termed AgNORs or ‘silver-staining nucleolar organiser regions’) although what is actually stained are specific regions of the chromosomes concerned with nucleolar function. Nucleoli are especially prominent – and may be multiple – and AgNOR staining is particularly abnormal in malignant transformed cells. Severe clumping and fragmentation of chromatin together with nuclear shrinkage and break-up is suggestive of cell death and is characteristic of apoptosis.
The past 20 years have witnessed a revolution in human pathology, with the development of a wide range of antibodies that can be used for immunohistochemical studies on formalin-fixed and paraffin-embedded tissues. Consequently, with certain exceptions (most notably renal pathology), electron microscopy is rarely undertaken to study tissues in clinical histopathological practice. However, at higher magnification in the transmission electron microscope, fine indicators of cell damage can be seen earlier than those seen on ordinary light microscopy, but they are not much more specific. The general effects of loss of transmembrane ion and water control leads to swollen cells and swelling of mitochondria, both dependent upon the loss of ability to exclude calcium from the cell and from the mitochondrion. Smooth endoplasmic reticulum is dilated, and the ribosomes fall off the rough endoplasmic reticulum. Nuclear changes are similar to, but more pronounced than, those seen at light microscopy.
If a late step in a non-branching metabolic pathway is defective, either genetically or because of some form of trauma, then intermediates earlier in the pathway will accumulate. In some cases where there is branching of the pathway the accumulating materials may be diverted off into alternative processes and the end effect of the insult will be a loss of the usual products occurring after the defective step. Accumulations may be relatively inert, such as lipids occurring in the liver as described above, and their only significance may be as markers of damage. In other cases the accumulated materials may have deleterious effects resulting from direct metabolic influences, e.g. acidosis due to accumulated lactate, or by simple bulk effects such as those seen in various lysosomal storage diseases. Exogenous compounds may be metabolised or stored, but both of these processes may have deleterious consequences. Substances such as carbon tetrachloride are themselves not toxic, but the body has a limited and stereotyped series of responses to external agents and, whilst these responses are on the whole effective at detoxification, in some instances they can result in the production of molecular species more toxic than the original ingested material. In this manner carbon tetrachloride is metabolised in the liver with the production of free radicals which cause severe damage. A similar phenomenon is seen following paracetamol (acetaminophen) overdose. The paracetamol itself is not hepatotoxic, but it is metabolized to n-acetyl p-benzoquinonamine which is potentially hepatotoxic if glutathione levels are depleted. This can be inferred histologically since the liver damage does not occur around the portal vein branches where the carbon tetrachloride or paracetamol enters the liver but only at some distance from this in zones II and III as it becomes metabolised. In the case of ingested asbestos or silica particles, these are taken up into macrophages and cause the disruption of lysosomes, with the release of hydrolytic enzymes. There is consequent minute scarring from this single cell event, but the fibres are then taken up into another macrophage and the process is repeated. Some materials are totally inert, such as carbon, and serve only to show that the individual has a history of exposure to this substance and, more importantly, perhaps to other substances.
This is a group of extracellular proteins that accumulate in many different conditions and cause problems by a simple bulk effect. The precise composition of the amyloid is dependent upon the causative disease process. It accumulates around vessels and in general causes problems by progressive vascular occlusion. The common feature of all the conditions underlying amyloidosis is the production of large amounts of active proteins. These proteins are inactivated by transformation of their physical form into beta-pleated sheets which are inert (silk is a beta-pleated sheet, which is why silk sutures are not metabolised in the human body). The human body has no enzymes for metabolising beta-pleated sheets, and amyloid, therefore, accumulates. The material is waxy in appearance and reacts with iodine to form a blue-black pigment similar to the product of reaction of starch and iodine (amyloid = starch-like). The disparate origins of the proteins constituting amyloid can be demonstrated, as the proteins often retain some of their immunohistochemical properties. The rationale of this process is that it removes excess metabolically active circulating proteins and stores them in an inert form, which is advantageous if the cause is short-lived but can be deleterious if the condition causing the protein production continues. The types of disease associated with amyloid production are: chronic inflammatory processes such as tuberculosis, rheumatoid disease and chronic osteomyelitis; tumours with a large production of protein, typically myeloma; and miscellaneous disease with protein production such as some inflammatory skin diseases, some tumours of endocrine glands and neurodegenerative diseases such as Alzheimer’s disease.
Pigments of various sorts accumulate in cells and tissues. They may be endogenous or exogenous in origin and they represent a random collection of processes linked only by the fact that the materials happen to be coloured. When blood escapes from vessels into tissue the haemoglobin gives a dark grey-black colour to the bruise. As the haemoglobin is metabolised through biliverdin and bilirubin, it changes from green to yellow and is finally removed. Such haematomas generally have no significance unless they are very bulky or if they become infected. Other endogenous pigments include the bile pigments in obstructive jaundice. These can be seen in the skin and even more clearly in the sclera because they bind preferentially to elastin and this material occurs in greatest concentration in these tissues. Related pigments are found in the tissues in the porphyrias, but these absorb ultraviolet light and are not visibly coloured; however, they can transform this absorbed radiant energy into chemical energy, setting off free radical damage. Another pigment, beta-carotene, can be used in some porphyrias (erythropoietic protoporphyria) to quench free radical activity.
The commonest pigment in human skin is melanin, which is red/yellow (pheomelanin), or brown/black (eumelanin), but if it occurs in deep sites, as in blue naevi, can appear blue due to the Tindall effect. Melanin pigments do no harm, but they are often markers of pigmented tumour pathology. In widespread malignant melanoma the melanin production can be so great that melanin appears in the urine. Melanin production is under hormonal control, and ACTH, which is structurally related to MSH (melanocyte stimulating hormone), can cause pigmentation in situations in which it is produced in pathological amounts or iatrogenically. Melanosis coli is a heavy black pigmentation of the colon associated with anthracene laxative use and is unrelated to melanin – the pigment in melanosis coli is lipofuscin – and is itself inert. Melanin can be distinguished from haemosiderin and lipofuscin by its positive staining with the Masson Fontana method.
Haemosiderin is a granular light brown pigment composed of iron oxide and protein. It accumulates in tissues – particularly in the liver, pancreas, skin and gonads – in conditions where there is iron excess, either due to a genetic defect or iatrogenic administration. Haemosiderin also accumulates in tissues where bleeding has occurred. As the blood is broken down, the iron is phagocytosed by macrophages which become haemosiderin-laden. Haemosiderin can be distinguished from melanin and lipofuscin by its positive Prussian blue reaction when exposed to potassium ferrocyanide and hydrochloric acid.
Lipofuscin is a brown pigment that accumulates in ageing cells and is often called age pigment. It does not appear to cause any damage and is an incidental marker of ageing. It is mainly formed from old cellular membranes by the peroxidation of lipids which have become cross linked as a result of free radical damage and which accumulate in residual bodies without being further metabolised. They are thought to be mainly of mitochondrial origin. Lipofuscin shows neither the Prussian blue reaction nor is it stained with the Masson Fontana method.
Exogenous pigments are introduced in tattooing and some have been toxic in various ways. Mercuric chloride (a red pigment) and potassium dichromate (a green pigment) are commonly used in tattooing. Another source for exogenous pigmentation is drugs and organic halogen compounds have often been implicated in abnormal pigmentation problems.
These are another heterogeneous group of conditions, most of which affect joints, producing gout in the case of sodium urate crystals and pseudogout in the case of calcium pyrophosphate. Calcium oxalate crystals are commonly found within the colloid of normal thyroid tissue and may be associated with a low functional state of the thyroid follicles.
This occurs in two main pathological situations as well as physiologically in developing or healing bone: it occurs in normal tissues in the presence of high circulating levels of calcium ions (metastatic calcification) and in pathological tissue in the presence of normal serum levels of calcium (dystrophic calcification). Most calcium deposits are calcium phosphate in the form of hydroxyapatite and contain small amounts of iron and magnesium and other mineral salts.
Calcification occurs in two stages: initiation and propagation. Intracellular calcification begins in mitochondria, and in this context it is interesting to note that the earliest indicator of cell death is the influx of calcium into mitochondria. Extracellular initiation of calcification begins in small, membrane-bound matrix vesicles which seem to be derived from damaged or ageing cell membranes. They accumulate calcium and also appear to have phosphatases in them which release phosphate which binds the free calcium. Propagation is by subsequent crystal deposition which may be affected by a lowering of calcification inhibitors and the presence of free collagen.
This term can be used to refer to the whole range of agents that can damage cells, tissues or organisms, but is commonly restricted to mechanical damage. It is often lumped together with other non-chemical, non-biological forms of damage under the heading of physical damage, which includes extremes of temperature and the various forms of radiation.
Mechanical damage is seldom so specific that it acts only at the individual cellular level – such damage usually involves at least groups of adjacent cells – but laser techniques make it possible to study individual cell damage. If cells are damaged in this way they appear to be able to ‘clot’ small areas of cytoplasm and then to heal this by secreting new cell membrane.
Freezing cells slowly produces ice crystals which act as ‘micro-knives’ cutting macromolecules as they grow. Cryotechniques require very rapid freezing to prevent ice crystal formation, sometimes in conjunction with chemicals which inhibit crystal formation.
Heating cells introduces free energy and causes macromolecules to vibrate and break. Various intracellular mechanisms are present to repair these breaks, but there is a critical level at which cells are overwhelmed and death ensues. Enzymes have a temperature optimum at which their catalytic rate is maximum, and body temperature is carefully maintained in mammals and birds so that enzymes work close to this optimum. The optimum is not necessarily the maximum rate, and metabolism speeds up as temperature rises, so that fever states are catabolic. In some cases it seems that the body’s thermostat is deliberately reset at a higher level in an effort to deal with various infections, the causative organisms of which are even more temperature sensitive.
This may be in the form of electromagnetic waves or particles and also introduces free energy into cells. The longer the wavelength the lower the energy of the radiation. At very low wavelengths we are back in the realms of simple heat. In the case of radiation we have the added problem of iatrogenic damage since many medical activities involve exposing the patient to some form of radiation, including both diagnostic and therapeutic modalities. Most types of radiation used in medicine cause the formation of free ions; they are consequently lumped together as ionising radiation.
The problem of variation in energy level of radiation has led to considerable difficulty in establishing suitable measures of dose. The favoured unit currently is the gray (Gy) which is a unit of absorbed dose. One gray is equivalent to 100 rad (the older dose unit of radiation absorbed dose). However, since radiations are often mixed and since tissues have different sensitivities, a mathematically corrected dose called the effective dose equivalent is now used, and the unit of this is the sievert (Sv). The environment contains a number of sources of natural radiation and some degree of contaminant radiation. These include radon liberated from uranium naturally occurring in granite bedrock, and cosmic radiation. The background radiation varies from area to area and with occupations. For example, those frequently engaged in air travel have a higher exposure to cosmic radiation, to which there is approximately a 100 times greater exposure at commercial flight altitudes than at sea level. A pilot flying 600–800 hours per year is exposed to approximately twice the background radiation dose −5 mSv/year – of someone who spends the year at sea level, which is approximately 2.5 mSv/year in the UK. There is considerable debate as to what constitutes a safe level of background radiation or even if there is such a thing as a level of radiation below which no damage will occur. It seems reasonable to assume that no level of radiation can be considered safe no matter how low it is since the safety is only a statistical statement of the likelihood of a mutational event and the probability can never be zero.
When radiation enters a cell it can be absorbed by macromolecules directly but more commonly it reacts with water to produce free radicals which then interact with macromolecules such as proteins and DNA. Both enzymatic and structural proteins depend on their three-dimensional (3-D) structure for their function, and this 3-D structure is dependent upon various types of chemical bonds. These bonds are disrupted by radiation, mostly by the intermediation of free radicals, and the proteins are then incapable of performing their structural or enzymatic duties. Radiation-induced DNA damage includes:
Various tissues differ in their susceptibility to radiation, but in general the most rapidly dividing tissues – the bone marrow and the epithelium of the gut – are the most sensitive. Radiation damage to tissues is generally divided into acute and chronic effects, but the precise effects at any time are strongly dose related. Acute effects are related to cell death and are most marked in those cells that are generally dividing rapidly to replace physiological cell loss such as gut epithelium, bone marrow, gonads and skin. DNA damage leads to an arrest of the cell cycle at the end of the G1-phase, due to the action of p53. If the damage cannot be repaired, apoptotic pathways (see below) are triggered. Damage is also due to vascular fragility as a result of endothelial damage. The chronic effects of radiation include atrophy which may be due to a reduction in cell replication combined with fibrosis. The initial insult may be vascular endothelial cell loss with exposure of the underlying collagen with subsequent platelet adherence and thrombosis. This is then incorporated into the vessel wall and is associated with intimal proliferation of endarteritis obliterans. Narrowing of the vessels due to endarteritis obliterans leads to long-term vascular insufficiency and consequent atrophy and fibrosis.
The effect of radiation is to suspend renewal of all cell lines. Granulocytes are reduced before erythrocytes, which survive much longer. The ultimate outcome depends on the dose used and the speed of delivery and varies from complete recovery to aplastic anaemia and death. In the long-term survivor there is an increased incidence of leukaemia.
Irradiation of the epidermis results in cessation of mitosis with desquamation and hair loss. If enough stem cells survive, hair will regrow and any epidermal defects will regenerate. Damage to melanocytes results in melanin deposition in the dermis, where it is ingested by phagocytic cells which remain in the skin and result in hyperpigmentation. Destruction of dermal fibroblasts results in an inability to produce collagen and subsequently to thinning of the dermis. Damage to small vessels in the skin is followed by thinning of their walls, with dilatation and tortuosity, and hence telangiectasia. Larger vessels undergo endarteritis obliterans with time.