Introduction

In aerobically growing eukaryotic cells, ATP synthesis is coupled to the enzymatic reduction of molecular oxygen (O2) to H2O via a sequential, four-electron transfer reaction. However, one- and two-electron reduction of O2 can also occur under physiological conditions in mitochondria and other cellular compartments, generating superoxide anion free radicals (O2−) and other active oxygen species, including hydrogen peroxide (H2O2) and the hydroxyl radical (OH) (Fridovich, 1983). Although several intracellular mechanisms exist that normally prevent the accumulation of active oxygen species during cell metabolism, these protective systems can become overwhelmed and toxic damage to cells may follow. Thus, accumulating evidence implicates oxygen radicals and other oxygen-derived species as causative agents in ageing and a variety of human diseases, including cancer (Cerutti, 1985).

The term ‘oxidative stress’ is generally applied to conditions in which the intracellular prooxidant–antioxidant ratio favours the former (Sies, 1985). Recent studies by our laboratory and others have examined the effects of oxidative stress on various cellular functions. Experimental systems commonly used to expose various types of cells and tissues to oxidative stress include enzymatic generation of active oxygen species (e.g. using xanthine/xanthine oxidase) (Muehlematter, Larsson & Cerutti, 1988), direct exposure to peroxides or prooxidants (Sies, Brigelius & Graf, 1987) and intracellular generation of active oxygen species by treatment with redox-cycling chemicals such as quinones or bipyridilium compounds (Smith et al., 1985; Orrenius et al., 1986).

Introduction

Malignant hyperthermia (MH) is an inherited disorder which predisposes sufferers to skeletal muscle hypercontraction, severe metabolic acidosis and a potentially lethal hyperthermia (Britt, 1985; McGrath, 1986; Sessler, 1986; O'Brien, 1987; Gronert, Mott & Lee, 1988; Harriman, 1988; Heffron, 1988; Rosenberg, 1988). In man, MH is usually triggered in susceptible individuals by halothane anaesthesia. A similar condition occurring in pigs is called the porcine stress syndrome (PSS). In response to halothane such PSS-susceptible pigs quickly develop a tachycardia and hyperventilate. Cyanotic areas then form on the skin, muscles hypercontract, limb rigidity occurs and a fatal hyperthermia ensues. Postmortem reveals oedematous, malodourous muscle with a severely disrupted structure. This meat is termed pale soft exudative (PSE) and cannot be used commercially. As well as halothane, stresses caused by transportation, exercise, feeding, mating and parturition will cause the development of a MH attack in pigs (Mitchell & Heffron, 1982).

PSS is regarded as a good model for human MH (Gronert, 1980) and has been used for much of the research into the mechanisms and causes of the disease. There is no uncomplicated and reliable diagnostic test for MH/PSS. Induction of limb rigidity by halothane inhalation will identify PSS-susceptible individuals and cause only limited mortality when performed by a skilled operator (Webb, 1980). In humans the disease is often recognised in a family when one person undergoes halothane anaesthesia prior to a surgical procedure. Thereafter muscle samples from other members of the family can be subjected to in vitro contracture tests to identify abnormal reaction to either halothane or caffeine.

Ultrastructural changes in muscle mitochondria during cell damage

During rapid cellular damage, the organelles frequently undergo major ultrastructural pathological changes which are shown most dramatically in the mitochondria. In particular, the mitochondria undergo apparent septation and subdivision, this phenomenon being most commonly seen in skeletal and cardiac muscle cells (Duncan, 1988). Are these ultrastructural changes in muscle mitochondria perhaps triggered by changes in the intracellular concentration of Ca2+ ([Ca2+]i) or by active oxygen radicals?

Lipid bodies, lipofuscin granules and myelin-like figures (or membrane whorls) are regarded as late-stage products of lysosomal digestion or as lysosome-derived elements in mammalian skeletal muscle; they occur rarely in human healthy muscle but are much more common in diseased muscle and in old age (Mastaglia & Walton, 1982; Dubowitz, 1985; Walton, 1988). Lipid droplets are present in many cells and are considered to lack a limiting membrane (Threadgold, 1976), but lipid bodies with a bounding membrane are clearly evident in the electronmicrographs of skeletal muscles of neonatal kittens (Tomanek, 1976), rats (Nag & Cheng, 1982) and dystrophic hamsters (Caulfield, 1966) as well as in ischaemic canine muscle (Stenger et al., 1962). Ultrastructural studies of mammalian skeletal muscle undergoing rapid cellular damage that is experimentally-induced in vitro, with a time-course of minutes (Duncan, 1988), show that lipid bodies develop quickly in association with the muscle mitochondria.

Introduction

Ischaemia encompasses a wide range of clinical conditions and is also an integral part of many surgical techniques, in particular transplantation. Organ retrieval usually involves a short period of warm ischaemia (WI) between cessation of the blood supply and harvesting the organ from the donor. This is followed by a much longer period of cold ischaemia (CI) in which the organ is flushed with and suspended in a cold asanguinous solution for transport to the recipient. The organs are then rapidly reperfused with fully oxygenated blood as soon as the vascular pedicle is reconstructed. Cooling depresses metabolism and very much slows the deterioration of ischaemic organs. However, some organs are particularly susceptible to ischaemic damage and it is currently considered inadvisable to store liver, heart or lungs for longer than 4 hr. Kidneys are usually stored for about 24 hr but storage periods up to 72 hr are not uncommon. There is no definitive safe storage time but rather the longer the period of ischaemia, the less chance there is of an organ functioning immediately upon transplantation. Acute renal failure may occur in transplanted kidneys which become enlarged with a pale cortex and a dark congested medulla and have a drastically impaired excretory capacity. Vascular injury is another possible complication in ischaemically damaged kidneys which are slow to perfuse when revascularised and develop a microagulopathy which results in an outflow block and venous stasis.

Introduction

Reperfusion of the ischaemic myocardium

The occlusion of a coronary artery leads to the development of myocardial ischaemia and initiates the process of infarction. It is clear that the readmission of blood to severely ischaemic tissue is essential if that tissue is ultimately to survive. Clinically, reperfusion can be achieved within hours of the onset of an ischaemic episode (using angioplasty and thrombolytic techniques), following prolonged ischaemia (using coronary artery bypass grafts) or may occur spontaneously following coronary artery spasm. The recent development of safe angioplasty techniques and effective thrombolytic drugs has led to an increased interest in the consequences of reperfusion. Although clinically such interventions can preserve myocardial function and improve prognosis (Serruys et al., 1986), there is a growing body of evidence from animal models to suggest that reperfusion can, in itself, promote a variety of undesirable effects such as arrhythmias (Manning & Hearse, 1984), myocardial stunning (Braunwald & Kloner, 1982), and leucocyte infiltration and vasoconstriction (Engler et al., 1986).

The concept that reperfusion of reversibly damaged but viable cells can promote lethal injury in that tissue is, however, controversial (Hearse, 1989; 1991; Opie, 1989). Although there is considerable experimental evidence implying that reperfusion may accelerate the expression of injury in cells that are ultimately destined to die, conclusive evidence that reperfusion itself can kill viable cells does not yet exist. The acceleration of tissue injury by reperfusion may, however, alter the processes underlying cell death and may therefore lead to differences in scar formation, leucocyte infiltration and susceptibility to aneurism. There is considerable evidence in support of the existence of this type of reperfusion-induced injury.

Introduction

Skeletal muscles are subjected to considerable physical stresses during normal contractile activity, and during excessive or unaccustomed exercise may become seriously damaged such that normal contractile function is impaired. This is evidenced by morphological and ultrastructural changes in muscle together with leakage of large intracellular components (such as certain cytosolic enzymes) into the extracellular fluid. Analogous changes appear to occur in various disease states such as the muscular dystrophies, malignant hyperthermia and various inflammatory myopathies.

In man, muscle damage can be conveniently monitored by measurement of the activity of various muscle-derived enzymes in the blood. The most commonly used of these are creatine kinase or aldolase with creatine kinase determination being particularly useful because of its high sensitivity and because analysis of the isoform pattern of the MM type allows an examination of the elapsed time since the occurrence of an episode of muscle damage leading to enzyme efflux (Page et al., 1989). Further evidence for, or confirmation of, the occurrence of damage can readily be obtained by percutaneous biopsy of the affected muscle under local anaesthetic (Edwards, MacLennan & Jackson, 1989) followed by histological or electron microscopic examination of the tissue. Studies designed to elucidate the mechanisms by which skeletal muscle damage occurs following exercise or in disease states are rare in comparison to studies of the heart or other organs, but a number have been undertaken using different systems. Many of the human studies have (of necessity) been non-invasive and descriptive from which little information concerning basic mechanisms can be obtained, but many further data have been obtained from experiments with animal models in vivo and from in vitro studies of isolated skeletal muscle tissue.

The underlying events during rapid cellular damage have been studied in a variety of cells, particularly kidney, hepatocytes and muscle cells, although the initial lesions are very different, being genetic (e.g. Duchenne muscular dystrophy, see Jackson, McArdle & Edwards, this volume; or malignant hyperthermia, see Arthur & Duthie, this volume), or the response to toxic agents, or the result of endocrine dysfunction or a failure in metabolism. It has been suggested that there may be final common pathways, or that there may be a common central trigger. In particular, Ca2+ has been proposed as having a major role in initiating and regulating these events in cell death and toxic cell killing (Duncan, 1978; Schanne et al., 1979; Farber, 1981; Trump, Berezesky & Osornio- Vargas, 1981; Nayler, 1983; Orrenius et al., 1989) and consequently these studies have concentrated on the ways in which the initial lesion may result in changes in the intracellular concentration of free Ca2+ ([Ca2+]i) and on the pathways that may be activated by a rise in [Ca2+]i. For example, the missing gene product (dystrophin) in Duchenne muscular dystrophy and in mdx mice has been identified as a sub-sarcolemma cytoskeletal protein and it remains to be explained how its absence leads to the rise in [Ca2+]i that has been measured, and to the breakdown of the sarcolemma myofilament apparatus (see Duncan, 1989a). There is little doubt that a rise in [Ca2+]i can precipitate rapid cell damage; it can be produced experimentally by exposure of cells to the divalent cation ionophore A23187 and an elevated [Ca2+]i has been measured in such conditions as malignant hyperthermia (Lopez et al., 1985).

Polymorphonuclear leukocytes (neutrophils) provide the first line of defence to protect the host against most bacterial and many fungal pathogens, and hence possess an array of specialised cytotoxic processes and associated pathways in order to perform this important function during phagocytosis. In the 1930s the importance of O2 was recognised when it was discovered (Baldridge & Gerard, 1933) that a ‘respiratory burst’ accompanied phagocytosis, but this was mistakenly believed to be due to increased mitochondrial respiration necessary to supply the extra energy required for phagocytosis. The unusual nature of the respiratory burst was not appreciated until later (Sbarra & Karnovsky, 1959) when it was found to be uninhibited by cyanide and hence not associated with mitochondrial respiration. Later, it was proposed that H2O2 was generated during phagocytosis (Iyer, Islam & Quastel, 1961) and that O2− was the primary product of O2 reduction (Babior, Kipnes & Curnutte, 1973). It was therefore suggested that the respiratory burst was required for the generation of oxygen metabolites which were instrumental in pathogen killing (Selvaraj & Sbarra, 1966). The link between the products of the burst and microbial killing was confirmed when it was discovered that phagocytes from patients with chronic granulomatous disease (CGD, formerly Fatal Granulomatous Disease of Childhood) who are predisposed to life-threatening infections had an impaired ability of their phagocytes to mount a respiratory burst (Holmes, Page & Good, 1967). The search for the molecular defects responsible for this condition (or group of related conditions) has proved invaluable in identifying the molecular components required for oxidant generation (Segal, 1989a,b), and also in elucidating the complex processes by which these become assembled and activated during phagocytosis.

Chapter 1 reviewed the biology of human skin pigmentation, and it is now appropriate to consider the pathology of the melanin pigmentary system and, if possible, to clarify the mechanisms producing these abnormalities. Basically, disturbances in human pigmentation manifest clinically as either excessive pigmentation (hyperpigmentation) or deficient pigmentation (hypopigmentation). Any respectable textbook of dermatology will provide lists of the legion conditions which fall under the rubric of the hyperpigmentation and hypopigmentation disorders respectively. Most of these are rare and of no interest to the general reader. This chapter will discuss some selected examples of the hyperpigmentation disorders and the following chapter will consider certain conditions associated with hypopigmentation.

It must be emphasized at the outset that the diagnosis of hyperpigmentation may be difficult. The normal skin colour of a Caucasoid of Mediterranean origin, for example, may not differ in the intensity of its hue from the abnormal pigmentation of a fair-skinned Scandinavian patient. Furthermore, pigmentation of the oral mucosa (e.g. gums) is usually pathological in fair-skinned Caucasoids but not in the darker ethnic groups (see p. 76).

Hormonal and metabolic factors

Reference was made in Chapter 2 to hyperpigmentation caused by the sex hormones (oestrogens and progesterone) and particularly to the chloasma induced by pregnancy and oral contraceptive agents (see Fig. 2.2).

The classic pathological condition causing hyperpigmentation is Addison's disease. This disease, described by Addison in 1855, is due to a failure of the adrenal glands to produce sufficient quantities of the adrenal hormones (corticosteroids). A common cause (and probably the commonest in Third World countries) is tuberculosis of the adrenal glands. Addison's disease results in weakness, lassitude and low blood pressure.

Albinism

Albinism consists of a group of genetic disorders of the melanin pigmentary system which occurs throughout the animal kingdom from insects, fish and birds right up to human beings. It is characterized by an absence of or decrease in melanin which, in the human varieties of albinism, takes two forms: oculocutaneous albinism and ocular albinism. The former (which is by far the commoner) manifests as a lack of pigmentation in the skin, hair and eyes; in ocular albinism the loss of melanin is limited to the eyes and skin pigmentation is normal. All human albinos have visual problems – there is hypopigmentation of the iris, choroid and retina as well as maldevelopment of the fovea, a part of the retina which mediates central vision. The typical eye signs are photophobia (an abnormal, often painful, sensitivity to sunlight leading to its avoidance), nystagmus (involuntary, rhythmical oscillations of the eyeballs, usually in a horizontal plane), squint and a decreased visual acuity (in severe cases amounting to partial blindness). This chapter will concern itself only with oculocutaneous albinism.

History

Allusions to albinism date from antiquity but the actual term ‘albino’ (from the Latin albus, white) was coined by the seventeenth-century Portuguese explorer, Balthazer Tellez, who sighted certain ‘white’ Negroids on the west coast of Africa. Columbus, however, was claimed to have encountered such people (near Trinidad) at the time of his fourth voyage to America in 1502. The identification of albinos was hardly a feat of recognition: compared with normally pigmented Negroids, these albinos were highly conspicuous, and it was noted that their marked photophobia confined them to their huts until twilight.