Hamilton, A., “ Industrial poisoning by compounds of the aromatic series.” J. Ind. Hygi. 200–212 (1919).
Hancock, G., A. E. Moffitt, Jr., and E. B. Hay, “Hematological findings among workers exposed to benzene at a
coke oven by-product recovery facility,” Arch. Environ. Health 39(6): 414–418 (1984).
Kipen, H. M., R. P. Cody, K. S. Crump, B. C. Allen, and B. D. Goldstein, “ Hematological effects of benzene: A
thirty-five year longitudinal study of rubber workers,” Toxicol. Ind. Health 4: 411–430 (1988).
Peterson, J. E., and R. D. Stewart, “Absorption and elimination of carbon monoxide by inactive young men.” Arch.
Environ. Health 21: 165–171 (1970).
Rinsky, R. A., A. B. Smith, R. Hornung, T. G. Filloon, R. J. Young, A. H. Okun, and P. J. Landrigan, “ Benzene and
Leukemia. An epidemiologic risk assessment,” N. Engl. J. Med. 316: 1044–1050 (1987).
Stewart, R. D., “The effects of low concentrations of carbon monoxide in man,” Scand. J. Respir. Dis. Suppl. 91:
56–62 (1974).
Yin, S N., Q. Li, Y. Liu, F. Tian, C. Du, and C. Jin. “Occupational exposure to benzene in China,” Br. J. Ind. Med.
44: 192–195 (1987).
REFERENCES AND SUGGESTED READING
109
5
Hepatotoxicity: Toxic Effects on the
Liver
HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
STEPHEN M. ROBERTS, ROBERT C. JAMES, AND MICHAEL R. FRANKLIN
This chapter will familiarize the reader with
•
The basis of liver injury
•
Normal liver functions
•
The role the liver plays in certain chemical-induced toxicities
•
Types of liver injury
•
Evaluation of liver injury

, Edited by Phillip L. Williams,
Robert C. James, and Stephen M. Roberts.
ISBN 0-471-29321-0 © 2000 John Wiley & Sons, Inc.
encountered by a drug or chemical after absorption from the gastrointestinal tract or peritoneal space
also means that the liver often sees potential toxicants at their highest concentrations. The same drug
or chemical at the same dose absorbed from the lungs or through the skin, for example, may be less
toxic to the liver because the concentrations in blood reaching the liver are lower, from both dilution
and distribution to other organs and tissues.
Figure 5.1 The liver maintains a unique position within the circulatory system.
112
HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
A second reason for the susceptibility of the liver to chemical attack is that it is the primary organ
for the biotransformation of chemicals within the body. As discussed in Chapter 3, the desired net
outcome of the biotransformation process is generally to alter the chemical in such a way that it is (1)
no longer biologically active within the body and (2) more polar and water-soluble and, consequently,
more easily excreted from the body. Thus, in most instances, the liver acts as a
detoxification
organ. It
lowers the biological activity and blood concentrations of a chemical that might otherwise accumulate
to toxic levels within the body. For example, it has been estimated that the time required to excrete
one-half of a single dose of benzene would be about 100 years if the liver did not metabolize it. The
primary disadvantage of the liver’s role as the main organ metabolizing chemicals, however, is that
toxic reactive chemicals or short-lived intermediates can be formed during the biotransformation
process. Of course, the liver, as the site of formation of these bioactivated forms of the chemical, usually
receives the brunt of their effects.
Morphologic Considerations
The liver can be described as a large mass of cells packed around vascular trees of arteries and veins
(see Figure 5.2). Blood supply to the liver comes from the hepatic artery and the portal vein, the former
normally supplying about 20 percent of blood reaching the liver and the latter about 80 percent.
Terminal branches of the hepatic artery and portal vein are found together with the bile duct (Figure

periportal
,
while those near the hepatic venule are termed
perivenular
. The hepatic venule is visualized as
occupying the center of the lobule, and cells surrounding the venule are sometimes termed
centrilobu-
lar
, while those farther away, near the portal triad, are called
peripheral

lobular
. Rappaport proposed
a different view of hepatic anatomy in which the basic anatomical unit is called the
simple liver acinus
.
In this view (Figure 5.3, left), cells within the acinus are divided into zones. The area adjacent to small
vessels radiating from the portal triad is zone 1. Cells in zone 1 are first to receive blood through the
sinusoids. Blood then travels past cells in zones 2 and 3 before reaching the hepatic venule. As can be
seen in Figure 5.3, zone 3 is roughly analogous to the centrilobular region of the classic lobule, since
it is closest to the central vein. Zone 3 cells from adjacent acini form a star-shaped pattern around this
vessel. Zone 1 cells surround the terminal afferent branches of the portal vein and hepatic artery, and
are often stated as occupying the
periportal
region, while cells between zones 1 and 3 (i.e., in zone 2)
are said to occupy the
midzonal
region. A modification of the typical lobule and acinar models has
been provided by Lamers and colleagues (1989) (Figure 5.3, right). Based on histopathologic and
immunohistochemical studies, they propose that zone 3 should be viewed as a circular, rather than

. Although compara-
tively few in number, these cells play an important role in phagocytizing microorganisms and foreign
particulates in the blood. While these cells are a part of the liver, they are also part of the immune
Figure 5.4 Liver section from mouse given an hepatotoxic dose of acetaminophen. With acetaminophen, liver
cell swelling and death characteristically occurs in regions around the central vein (Zone 3, arrow); cells near the
portal triad (Zone 1, arrow head) are spared.
5.1 THE PHYSIOLOGIC AND MORPHOLOGIC BASES OF LIVER INJURY
115
system. They are capable of releasing reactive oxygen species and cytokines, and play an important
role in inflammatory responses in the liver. The liver also contains
Ito cells
(also termed
fat-storing
cells, parasinusoidal cells,
or
stellate cells
) which lie between parenchymal and endothelial cells. These
cells appear to be important in producing collagen and in vitamin A storage and metabolism.
5.2 TYPES OF LIVER INJURY
All chemicals do not produce the same type of liver injury. Rather, the type of lesion or effect observed
is dependent on the chemical involved, the dose, and the duration of exposure. Some types of injury
are the result of acute toxicity to the liver, while others appear only after chronic exposure or treatment.
Basic types of liver injury include the anomalies described in the following paragraphs.
Hepatocellular Degeneration and Death
Many hepatotoxicants are capable of injuring liver cells directly, leading to cellular degeneration and
death. A variety of organelles and structures within the liver cell can be affected by chemicals. Principal
targets include the following:
1.
Mitochondria.
These organelles are important for energy metabolism and synthesis of ATP.

Nucleus.
There are several ways in which the nuclei can be damaged by chemical toxicants.
Some chemicals or their metabolites can bind to DNA, producing mutations (see Chapter 12). These
mutations can alter critical functions of the cell leading to cell death, or can contribute to malignant
transformation of the cell to produce cancer. Some chemicals appear to cause activation of endonu-
cleases, enzymes located in the nucleus that digest chromatin material. This leads to uncontrolled
digestion of the cell’s DNA—obviously not conducive to normal cell functioning. Some chemicals
116
HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
cause disarrangement of chromatin material within the nucleus. Morphologically, damage to the
nucleus appears as alterations in the nuclear envelope, in chromatin structure, and in arrangement of
nucleoli. Examples of chemicals that produce nuclear alterations include aflatoxin B, beryllium,
ethionine, galactosamine, and nitrosamines.
5.
Lysosomes.
These subcellular structures contain digestive enzymes (e.g., proteases) and are
important in degrading damaged or aging cellular constituents. In hepatocytes injured by chemical
toxicants, their numbers and size are often increased. Typically, this is not because they are a direct
target for chemical attack, but rather reflects the response of the cell to the need to remove increased
levels of damaged cellular materials caused by the chemical.
Not all hepatocellular toxicity leads to cell death. Cells may display a variety of morphologic
abnormalities in response to chemical insult and still recover. These include cell swelling, dilated
endoplasmic reticulum, condensed mitochondria and chromatin material in the nucleus, and blebs on
the plasma membrane. More severe morphological changes are indicative that the cell will not recover,
and will proceed to cell death, that is, undergo
necrosis
. Examples of morphological signs of necrosis
are massive swelling of the cell, marked clumping of nuclear chromatin, extreme swelling of
mitochondria, breaks in the plasma membrane, and the formation of cell fragments.
Necrosis from hepatotoxic chemicals can occur within distinct zones in the liver, be distributed

apoptosis
, or programmed cell death. Apoptosis is a normal
physiological process used by the body to remove cells when they are no longer needed or have become
functionally abnormal. In apoptosis, the cell “ commits suicide” through activation of its endonu-
cleases, destroying its DNA. Apoptotic cells are morphologically distinct from cells undergoing
necrosis as described above. Unlike cells undergoing necrosis, which swell and release their cellular
contents, apoptotic cells generally retain plasma membrane integrity and shrink, resulting in condensed
cytoplasm and dense chromatin in the nucleus. There are normally few apoptotic cells in liver, but the
number may be increased in response to some hepatotoxic chemicals, notably thioacetamine and
ethanol. Also, some chemicals produce hypertrophy, or growth of the liver beyond its normal size.
5.2 TYPES OF LIVER INJURY
117
TABLE 5.1 Drugs and Chemicals that Produce Zonal Hepatic Necrosis
Chemical
Site of Necrosis
Zone1 Zone 2 Zone 3
Acetaminophen X
Aflatoxin X X
Allyl alcohol X
Alloxan X
α
-Amanitin X
Arsenic, inorganic X
Beryllium X
Botulinum toxin X
Bromobenzene X
Bromotrichoromethane X
Carbon tetrachloride X
Chlorobenzenes X
Chloroform X

Source:
Adapted from Cullen and Reubner, 1991.
a
Necrosis is shifted to zone 1 in phenobarbital-pretreated animals.
118
HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
Examples include lead nitrate and phenobarbital. When exposure or treatment with these agents has
ended, the liver will return to its normal size. During this phase, the number of apoptotic cells is
increased, reflecting an effort by the liver to reduce its size, in part by eliminating some of its cells.
Drugs and chemicals can produce hepatocellular degeneration and death by many possible
mechanisms. For some hepatotoxicants, the mechanism of toxicity is reasonably well established. For
example, galactosamine is thought to cause cell death by depleting uridine triphosphate, which is
essential for synthesis of membrane glycoproteins. For most hepatotoxicants, however, key biochemi-
cal effects responsible for hepatocellular necrosis remain uncertain. The search for a broadly applicable
mechanism of hepatotoxicity has yielded several candidates:
Lipid Peroxidation
Many hepatotoxicants generate free radicals in the liver. In some cases, such as
carbon tetrachloride, the free radicals are breakdown products of the chemical generated by its
cytochrome P450-mediated metabolism in the liver. In other cases, the chemical causes a disruption
in oxidative metabolism within the cell, leading to the generation of reactive oxygen species. An
important potential consequence of free-radical formation is the occurrence of lipid peroxidation in
membranes within the cell. Lipid peroxidation occurs when free radicals attack the unsaturated bonds
of fatty acids, particularly those in phospholipids. The free radical reacts with the fatty acid carbon
chain, abstracting a hydrogen. This causes a fatty acid carbon to become a radical, with rearrangement
of double bonds in the fatty acid carbon chain. This carbon radical in the fatty acid reacts with oxygen
in a series of steps to produce a lipid hydroperoxide and a lipid radical that can then react with another
fatty acid carbon. The peroxidation of the lipid becomes a chain reaction, resulting in fragmentation
and destruction of the lipid. Because of the importance of phospholipids in membrane structure, the
principal consequence of lipid peroxidation for the cell is loss of membrane function. The reactive
products generated by lipid peroxidation can interact with other components of the cell as well, and

N
-acetyl-
p
-
5.2 TYPES OF LIVER INJURY
119
benzoquinone imine, have been identified, none as yet has been clearly shown to be instrumental in
acetaminophen-induced hepatic necrosis. Without identification of the critical target(s) for irreversible
binding for hepatotoxicants, this remains an attractive but unproven mechanism.
Loss of Calcium Homeostasis
Intracellular calcium is important in regulating a variety of critical
intracellular processes, and the concentration of calcium within the cell is normally tightly regulated.
The plasma membrane actively extrudes calcium ion from the cell to maintain cytosolic concentrations
at a low level compared with the external environment (the ratio of intracellular to extracellular
concentration is about 1:10,000). Both the mitochondria and endoplasmic reticulum are capable of
sequestering and releasing calcium ion as needed to modulate calcium concentrations for normal cell
functioning. Loss of control of intracellular calcium can lead to a sustained rise in intracellular calcium
levels, which, in turn, disrupts mitochondrial metabolism and ATP synthesis, damages microfilaments
used to support cell structure, and activates degradative enzymes within the cell. These events could
easily account for cell death from hepatotoxic chemicals.
Early studies of toxic effects of chemicals on liver cells in culture suggested that an influx of calcium
from outside the cell (e.g., from plasma membrane failure) was responsible for their toxic effects. Later
experiments showed that this was probably not the case, but nonetheless supported disregulation of
intracellular calcium as a key event in toxicity. Intracellular calcium levels were observed to rise
substantially in response to a number of hepatotoxicants, apparently due to chemical effects on
mitochondria and/or the endoplasmic reticulum leading to loss of control of intracellular calcium
stores. Impaired extrusion of calcium out of the cell by the plasma membrane might also be important,
at least for some chemicals. In general, increases in intracellular calcium preceded losses of viability,
suggesting a cause–effect relationship. It is sometimes difficult, however, to discern to what extent
elevated calcium levels are the cause of, or merely the result of, cytotoxicity.

macrovesicular steatosis
) or numerous small vacuoles (
microvesicular steatosis
). The type of
steatosis (macro- or microvesicular) is characteristic of specific hepatotoxicants and, in some cases,
of certain diseases or conditions. For example, microvesicular steatosis has been associated with
120
HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
tetracycline, valproic acid, salicylates, aflatoxin, dimethylformamide, and some of the antiviral
nucleoside analogs used to treat HIV. It is also associated with Reye’s syndrome and fatty liver of
pregnancy. Macrovesicular steatosis has been associated with antimony, barium salts, carbon disulfide,
dichloroethylene, ethanol, hydrazine, methyl and ethyl bromide, thallium, and uranium compounds.
There are several potential chemical effects that can give rise to accumulation of lipids in the cell.
These include:
1.
Inhibition of Lipoprotein Synthesis.
A number of chemicals are capable of inhibiting synthesis
of the protein moiety needed for synthesis of lipoproteins in the liver. These include carbon
tetrachloride, ethionine, and puromycin.
2.
Decreased Conjugation of Triglycerides with Lipoproteins.
Another critical step in lipoprotein
synthesis is conjugation of the protein moiety with triglyceride. Carbon tetrachloride, for
example, can interfere with this step.
3.
Interference with Very-Low-Density Lipoprotein (VLDL) Transfer
. Inhibition of transfer of
VLDL out of the cell results in its accumulation. Tetracycline is an example of an agent that
interferes with this transfer.
4.

Dimethylhydrazine Tetracycline
Ethanol Thallium compounds
Ethionine Uranium compounds
Ethyl bromide White phosphorus
5.2 TYPES OF LIVER INJURY
121
“ foamy” cytoplasm. Often this condition progresses to cirrhosis. Examples of drugs associated with
phospholipidosis include amiodarone, chlorphentermine, and 4,4′-diethylaminoethoxyhexoestrol.
Cholestasis
The term
cholestasis
refers to decreased or arrested bile flow. Many drugs and chemicals are able to
produce cholestatic injury, and examples are listed in Table 5.3. There are several potential causes of
impaired bile flow, many of which can become the basis for drug- or chemical-induced cholestasis.
Some of these are related to loss of integrity of the canalicular system that collects bile and carries it
to the gall bladder, while others are related to the formation and secretion of bile. For example,
α-naphthylisothiocyanate disrupts the tight junctions between hepatocytes that help form the canali-
culi, the smallest vessels of the bile collection system. This causes a leakage of bile contents out of the
canaliculi into the sinusoids. Other toxicants, such as methylene dianiline and paraquat, impede bile
flow by damaging the bile ducts. The primary driving force for bile formation is the secretion of bile
acids into the canalicular lumen. This requires uptake of bile acids from the blood into hepatocytes,
and then transport into the canaliculus. Anabolic steroids are an example of a class of compounds that
produce cholestatic injury by inhibiting these transport processes.
Some cholestatic injury can be expected whenever there is severe hepatic injury of any type. This
is because normal bile flow requires functioning hepatocytes as well as a reasonably intact cellular
architecture in the liver. Whenever this is disrupted, some impairment of bile flow can be expected as
a secondary consequence. Many agents produce primarily hepatic necrosis with perhaps limited
cholestasis (see Table 5.1), others produce primarily cholestasis with some necrosis (chlorpromazine
and erythromycin are examples), and still others are capable of producing cholestasis with little or no
damage to the hepatocytes. The contraceptive and anabolic steroids are examples of this last category

this effect.
Peliosis hepatis
is another vascular lesion characterized by the presence of large, blood-filled
cavities. It is unclear why these cavities form, but there is reason to suspect that it may be due to a
weakening of sinusoidal supporting membranes. Use of anabolic steroids has been associated with this
effect. Although patients with peliosis hepatis are usually without symptoms, the cavities occasionally
rupture causing bleeding into the abdominal cavity.
Cirrhosis
Chronic liver injury often results in the accumulation of collagen fibers within the liver, leading to
fibrosis. Fibrotic tissue accumulates with repeated hepatic insult, making it difficult for the liver to
replace damaged cells and still maintain normal hepatic architecture. Fibrous tissue begins to form
walls separating cells. Distortions in hepatic microcirculation cause cells to become hypoxic and die,
leading to more fibrotic scar tissue. Ultimately, the organization of the liver is reduced to nodules of
regenerating hepatocytes surrounded by walls of fibrous tissue. This condition is called
cirrhosis
.
Hepatic cirrhosis is irreversible and carries with it substantial medical risks. Blood flow through the
liver becomes obstructed, leading to portal hypertension. To relieve this pressure, blood is diverted
past the liver through various shunts not well suited for this purpose. It is common for vessels associated
with these shunts to rupture, leading to internal hemorrhage. Even without hemorrhagic episodes, the
liver may continue to decline until hepatic failure occurs.
The ability of chronic ethanol ingestion to produce cirrhosis is widely appreciated. Occupational
exposures to carbon tetrachloride, trinitrotoluene, tetrachloroethane, and dimethylnitrosamine have
also been implicated as causing cirrhosis, as well as the medical use of arsenicals and methotrexate.
Some drugs (e.g., methyldopa, nitrofurantoin, isoniazid, diclofenac) produce an idiosyncratic reaction
resembling viral hepatitis. This condition, termed
chronic active hepatitis
, can also lead to cirrhosis if
the drug is not withdrawn.
Tumors

or glandlike structures are called
adenomas
, and in the liver these can occur among hepatocytes or bile
duct cells. Benign tumors of fibrotic cell origin are termed
fibromas
, and those in the bile ducts are
called
cholangiofibromas
.
5.2 TYPES OF LIVER INJURY
123
To make things more complicated, cells go through a series of morphological changes as they
progress to become a benign or malignant tumor. Thus, groups of cells that represent proliferation of
liver tissue, but are not (or not yet) tumors, may be described as nodular hyperplasia, focal hepatocel-
lular hyperplasia, or foci of hepatocellular alteration, depending on their morphological characteristics.
The foci of hepatocellular alteration represent the earliest stages that can be detected microscopically.
These foci are small groups of cells that are abnormal, but have no distinct boundary separating them
from adjacent cells. Their growth rate is such that they are producing little or no compression of
surrounding cells. The abnormalities are subtle at this stage, and special stains and markers are
sometimes used to help visualize them. Nodular hyperplasia is more readily observed; the group of
cells is more circumscribed and compression of adjacent cells is apparent. These cells are thought to
represent an intermediate step in tumor development. The significance of these lesions is not that they
are associated with any clinical signs or symptoms of disease, but rather that they may represent an
area from which a tumor may develop. Consequently, their appearance is important in the assessment
of the ability of a drug or chemical to cause cancer. For most chemicals, only a very small
percentage—or perhaps none—of the neoplastic areas will go on to produce a malignant tumor.
Consequently, the issue of how to use data regarding the appearance of these lesions in the assessment
of carcinogencity of a chemical is one of considerable discussion and debate among toxicologists.
Liver tumors from chemical exposure can arise through numerous mechanisms. Some hepatocar-
cinogens form DNA adducts leading to mutations. Nitrosoureas and nitrosamines are examples of

Pruritis
, or an itching sensation
in the skin, will often accompany the jaundice.
If the injury is particularly severe, it may lead to
fulminant hepatic failure
. When the liver fails,
death can occur in as little as 10 days. There are several complications associated with fulminant hepatic
124
HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
failure. Because the liver is no longer able to produce clotting factor proteins, albumin, or glucose,
hemorrhage and hypoglycemia are common. Also, failure of the liver leads to renal failure and
deterioration of the central nervous system (
hepatic encephalopathy
). Inability to sustain blood
pressure and accumulation of fluid in the lungs may also result. Prognosis is poor for patients with
fulminant hepatic failure, with a mortality rate of about 90 percent.
Morphologic Evaluation
For laboratory animal studies of hepatotoxicity, histopathologic examination of liver tissue by light or
electron microscopy can be extremely valuable. Histopathologic evaluation can provide information
on the nature of the lesion and the regions of the liver affected. This, in turn, can provide insight as to
the mechanism of toxicity. For example, the presence of fatty liver would suggest that the chemical
may interfere with triglyceride metabolism and/or lipoprotein secretion by the liver. Hepatocellular
necrosis confined to the centrilobular region might suggest bioactivation of the chemical by cytochrome
P450, since most of the activity of this enzyme normally exists in centrilobular cells. Altered
morphology of mitochondria as an early event in toxicity might suggest that mitochondrial toxicity is
an important initiating event in the sequence of events leading up to cell death. Histopathologic
observations alone cannot establish the mechanism of toxicity, and additional experimentation would
be required to explore these hypotheses. Nevertheless, morphologic observation provides important
clues, and is an integral part of any comprehensive study of potential hepatotoxicity of a chemical.
In humans, morphologic evaluation of liver biopsies is sometimes used in the diagnosis and

Serum Bilirubin.
The liver conjugates bilirubin, a normal breakdown product of the heme from
red blood cells, and secretes the glucuronide conjugate into the bile. Impairment of normal conjugation
5.3 EVALUATION OF LIVER INJURY
125
and excretion of bilirubin results in its accumulation in the blood, leading to jaundice. Serum bilirubin
concentrations may be elevated from acute hepatocellular injury, cholestatic injury, or biliary obstruc-
tion. This test is always included among the battery of tests to assess liver function clinically, although
it is not a particularly sensitive test for acute injury.
4.
Dye Clearance Tests.
These tests involve administration of a dye that is cleared by the liver and
measurement of its rate of disappearance from the blood. Delayed clearance is interpreted as evidence
of liver injury. One such dye is sulfobromophthalein (Bromsulphalein; or BSP). Clearance of BSP
from the blood is dependent on its active transport into liver cells, conjugation with glutathione, and
then active transport into the bile. Conceivably, disruption of any of these processes could result in
delayed clearance, although the biliary excretion step is regarded as most critical. The test consists of
administering a dose of the dye intravenously and measuring its concentration in blood spectro-
photometrically over time. Another dye used for this purpose is indocyanine green (ICG). Unlike BSP,
ICG is excreted into the bile without conjugation. Following an intravenous dose, the disappearance
of ICG from blood can be measured with repeated blood samples or noninvasively by ear densitometry.
The dye tests, although well established, are seldom used clinically.
5.
Drug Clearance Tests.
This test relies on the principle that liver injury will result in impaired
biotransformation. The biotransformation capacity of the liver is assessed by following the rate of
elimination of a test drug whose clearance from blood is dependent on hepatic metabolism (i.e., a drug
for which other elimination processes, such as renal excretion, are insignificant). A test drug such as
antipyrine, aminopyrine, or caffeine is administered, and its rate of disappearance from blood is
followed over time through serial blood sampling. This rate is compared with a value considered

′
-Nucleotidase 5
′
ND Increases reflect primarily cholestatic injury
Sorbitol dehydrogenase SDH High specificity for liver; increase reflects primarily
hepatocellular damage
Ornithine carbamoyltransferase OCT High specificity for liver; increase reflects primarily
hepatocellular damage
126
HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
assays that are rapid and inexpensive. In fact, the concentrations of each of these proteins are typically
measured as an enzyme activity rate, rather than a true concentration per se.
Aminotransferase activities [alanine aminotransferase (ALT) and aspartate aminotransferase
(AST)], alkaline phosphatase activity, and gamma glutamyltransferase transpeptidase (GGTP) are
included in nearly all standard clinical test suites to assess potential hepatotoxicity. The value of
performing a battery of these tests is that each test responds slightly differently in the various forms
of liver injury, and evaluating the pattern of responses can offer insight into the type of injury that has
occurred. For example, severe hepatic injury from acetaminophen can result in dramatic increases in
serum ALT and ALT activities (up to 500 times normal values), but only modest increases in alkaline
phosphatase activity. Pronounced increases in alkaline phosphatase is characteristic of cholestatic
injury, where increases in ALT and AST may be limited or nonexistent. In alcoholic liver disease, AST
activity is usually greater than ALT activity, but for most other forms of hepatocellular injury ALT
activities are higher. Serum GGTP is an extremely sensitive indicator of hepatobiliary effects, and may
be elevated simply by drinking alcoholic beverages. It is not a particularly specific indicator (it is
increased by both hepatocellular and cholestatic injury) and is best utilized in combination with other
tests. Serum levels of enzymes such as lactate dehydrogenase have been used to evaluate liver toxicity,
but this enzyme has such low specificity for the liver that interpretation of these results is impossible
without other confirming tests. Other enzymes such as sorbitol dehydrogenase (SDH) and ornithine
carbamoyltransferase (OCT) are quite specific to the liver.
5.4 SUMMARY


24
(1): 77–83 (1996).
REFERENCES AND SUGGESTED READING
127
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10: 72–76 (1989).
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Raton, FL, 1991, pp. 93–138.
MacSween, R. N. M., and R. J. Scothorne, “ Developmental anatomy and normal structure,” in Pathology of the
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Livingstone, Edinburgh, 1994, pp. 1–49.
Miyai, K., “Structural organization of the liver,” in Hepatotoxicity, R. G. Meeks, S. D. Harrison, and R. J. Bull,
eds., CRC Press, Boca Raton, FL, 1991, pp. 1–65.
Moslen, M. T., “ Toxic responses of the liver,” Casarett and Doull’s Toxicology. The Basic Science of Poisons, 5th
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Rappaport, A. M., “Physioanatomical basis of toxic liver injury,” in Toxic Injury of the Liver, Part A, E. Farber and
M. M. Fisher, eds., Marcel Dekker, New York, 1979, pp. 1–57.
Zimmerman, H. J., and K. G. Ishak, “Hepatic injury due to drugs and toxins,” in Pathology of the Liver, R. N. M.
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1994, pp. 563–633.
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HEPATOTOXICITY: TOXIC EFFECTS ON THE LIVER
6
Nephrotoxicity: Toxic Responses of the
Kidney

The cortex constitutes the major portion of the kidney and receives about 85 percent of the total renal
blood flow. Consequently, if a toxicant is delivered to the kidney in the blood, the cortex will be exposed
to a very high proportion.
Blood Flow to the Kidneys
The kidneys represent approximately 0.5 percent of the total body weight, or approximately 300 g in
a 70-kg human. Yet the kidneys receive just under 25 percent of the total cardiac output, which is about
1.2–1.3 L blood/min, or 400 mL/100 g tissue/min. The rate of blood flow through the kidneys is much
greater than through other very well perfused tissues, including brain, heart, and liver. If the normal
blood hematocrit (i.e., that proportion of blood that is red blood cells) is 0.45, then the normal renal
plasma flow is approximately 660 to 715 mL/min. Yet only 125 mL/min of the total plasma flow is
129
Principles of Toxicology: Environmental and Industrial Applications, Second Edition
, Edited by Phillip L. Williams,
Robert C. James, and Stephen M. Roberts.
ISBN 0-471-29321-0 © 2000 John Wiley & Sons, Inc.
actually filtered by the kidney. Of this, the kidney reabsorbs approximately 99 percent, resulting in a
urine formation rate of only about 1.2 mL/min. Thus, the kidneys, which are perfused at approximately
1 L/min, form urine at approximately 1 mL/min or 0.1 percent of the perfusion. Because of the high
volume of blood flow to the kidneys, a chemical in the blood is delivered to this organ in relatively
large quantities.
The kidney requires large amounts of metabolic energy to remove wastes from the blood by tubular
secretion and to return filtered nutrients back to the blood. Roughly 10 percent of the normal resting
oxygen consumption is needed for the maintenance of proper kidney function. Therefore, the kidney
is sensitive to agents, such as barbiturates, that induce
ischemia,
a lack of oxygen caused by a decrease
in blood flow. Acute intoxication by barbiturates induces severe hypotension (i.e., low blood pressure)
and shock. The severe decrease in blood pressure results in a decrease in filtration of the plasma,
resulting in a decrease (oliguria) or cessation (anuria) of urine formation. At an early stage this is called
pre–renal failure,

example, 75 percent bound to plasma proteins has an effective filterable concentration of 25
percent its total plasma concentration. Small amounts of protein, principally the albumins, which
are important chemical-binding proteins, may appear in the glomerular filtrate, but these are then
normally reabsorbed. The glomerular filter can be made more permeable in certain disease states
and by actions of certain nephrotoxicants. Both circumstances may result in the appearance of
protein in the urine (proteinuria). If damage to the glomerular element is severe, the result is a
loss of a large amount of the plasma proteins. If this occurs at a rate greater than the rate at which
the liver can synthesize the plasma proteins, the result will be hypoproteinemia (lower than normal
levels of proteins in the blood) and a concomitant edema due to the reduction in osmotic pressure.
This clinical picture is sometimes referred to as the
nephrotic syndrome.
However, transient but
significant proteinuria occurs normally after prolonged standing or strenuous exercise, so a single
measurement of high protein levels in the urine may not indicate kidney damage.
Nephron Tubules and Tubular Reabsorption
The tubular element of the nephron selectively reab-
sorbs 98–99 percent of the salts and water of the initial glomerular filtrate. The tubular element of the
Figure 6.2 Cortical and juxtamedullary nephrons. Enlargement of representative kidney section in Figure 6.1c.
(Based on B. Brenner and F. Rector, The Kidney, Saunders, Philadelphia, 1976.)
6.1 BASIC KIDNEY STRUCTURES AND FUNCTIONS
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nephron consists of the proximal tubule, the loop of Henle, the distal tubule, and the collecting duct
(see Figure 6.3). The proximal tubule consists of a proximal convoluted section (pars convoluta) and
a distal straight section (pars recta). Substances that are actively reabsorbed in the proximal tubule
include glucose, sodium, potassium, phosphate, amino acids, sulfate, and uric acid. Essentially all
amino acids and glucose are reabsorbed in the proximal tubule, and virtually none normally appear in
the urine. Agents toxic to the proximal tubule cause amino acids and glucose to appear in the urine
(aminoaciduria and glycosuria). Even though 250 g of glucose normally passes through the kidney
daily, no more than 100 mg is usually excreted in 24 h. However, glucose does appear in excess
quantities in the urine if high blood glucose levels produce a glucose load in the filtrate and this exceeds

also true. The result is that substances reabsorbed from the tubule will have a clearance significantly
less than the glomerular filtration rate (approximately 125 mL/min), while those secreted into the
tubules will have a clearance greater than the glomerular filtration rate in the adult human.
The Loop of Henle
After the glomerular filtrate has passed the proximal tubule in the nephron, it
moves into the loop of Henle. A nephron with a glomerulus in the outer portion of the renal cortex has
a short loop of Henle, whereas a nephron with a glomerulus close to the border between the cortex and
medulla (juxtamedullary nephrons) has a long loop of Henle extending into the medulla and papilla
(Figures 6.2 and 6.3). Approximately 15 percent of the nephrons in humans are juxtamedullary. As the
tubule descends into the medulla there is an increase in osmolality of the interstitial fluid. In the
descending limb the tubular fluid becomes hypertonic (high in salt) as water leaves the tubule to
maintain isoosmolality with the hypertonic interstitial fluid. However, in the thick segment of the
ascending portion of the loop of Henle the tubule becomes impermeable to water, and sodium is actively
transported out of the tubule with a decrease in the osmolality of the filtrate and an increase in the
osmolality of the interstitial fluid. The sodium transport in the ascending limb is necessary for
maintenance of the interstitial fluid concentration gradient. An additional 5 percent of the glomerular
filtrate fluid is reabsorbed in the loop of Henle, making a total of 80 percent of the total water reabsorbed
at this point.
Urine Formation
Once the tubular fluid enters the distal convoluted tubule and collecting duct, it is
hypotonic (low salt concentration) in comparison to blood plasma because of the active transport of
sodium out of the tubule at the loop of Henle. In the presence of vasopressin, the antidiuretic hormone,
the collecting duct becomes permeable to water, and the water moves from the tubular fluid in order
to maintain isoosmolality. However, in the absence of vasopressin, the collecting duct is impermeable
to water, which results in excretion of a large volume of hypotonic urine. Normally, another 19 percent
of the original glomerular filtrate fluid is reabsorbed in the last portion of the nephron, so that a total
of 99 percent of the fluid filtered at the glomerulus is reabsorbed—only 1 percent of the fluid entering
the nephron is excreted in the urine. Thus, the normal flow of urine is only about 1 mL/min, while in
the absence of vasopressin it can be increased to 16 mL/min. The kidney’s ability to concentrate urine
is determined by the measurement of urine osmolality. Urine osmolality can vary between 50 and 1400

Important Kidney Functions Seldom Considered as Toxic Endpoints
Renal Erythropoietic Factor
The kidney synthesizes hormones essential for certain metabolic func-
tions. For example, hypoxia stimulates the kidneys to secrete renal erythropoietic factor, which acts
on a blood globulin (proerythropoietin) released from the liver to form erythropoietin, a circulating
glycoprotein with a molecular weight of 60,000 daltons. The erythropoietin acts on erythropoietin-
sensitive stem cells in the bone marrow, stimulating them to increase hemoglobin synthesis, produce
more red blood cells, and release them into the circulating blood. The increased oxygen-carrying
capacity of the blood reduces the effects of hypoxia. Thus, in chronic renal failure, anemia usually
develops, in large part caused by decreased synthesis of erythropoietic factor because of damage to
the kidney tissues responsible for its synthesis. In addition to hypoxia, androgens and cobalt salts also
increase production of renal erythropoietic factor by the kidneys. In fact, administration of cobalt salts
produces an overabundance of red cells in the blood (i.e., polycythemia) by this mechanism. Poly-
cythemia has been observed in heavy drinkers of cobalt-contaminated beer.
Regulation of Blood Pressure
The kidney is involved in regulating blood pressure in several ways.
The kidney produces renin, a proteolytic enzyme, which cleaves a plasma protein globulin to form
angiotensin I. Angiotensin I is converted to angiotensin II, a potent vasoconstrictor. The angiotensin
II stimulates release of aldosterone from the adrenal cortex, and aldosterone increases reabsorption of
sodium in the kidney, leading to an increase in blood plasma osmolality and an increase in extracellular
volume. A decrease in the mean renal arterial pressure is the stimulus controlling kidney renin
production and the compensatory increase in arterial pressure by the abovementioned mechanisms. In
addition, renal disease and narrowing of the renal arteries are known to cause sustained hypertension
in humans. It appears that the kidney produces vasodepressor substances that are thought to be
important in the regulation of blood pressure. Thus, changes in the kidney that disturb the renin–
angiotensin–aldosterone system and/or secretion of the vasodepressor substances are suspected of
playing a key role in the etiology of certain forms of hypertension.
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NEPHROTOXICITY: TOXIC RESPONSES OF THE KIDNEY