Histopathology: Has a substance caused pathological changes
in body tissues?
Retinoids disrupt the ordered structure of bone and skin,
while calcium-channel blockers alter gum structure
Establishing the extent to which human or animal tissues
or organs are affected by the action of drugs is the task
of histopathology. The analyzed changes range from slight
abnormalities to pronounced injury. A suitable example for
demonstrating the damage caused to skin and bone is provided
by retinoids (vitamin A derivatives), substances used in the
treatment of acne. Microscopic investigations reveal that
healthy skin and its adnexa can react to such treatment by
becoming hyperplastic (increased organ volume as a result
of cellular multiplication). On the other hand, the same treatment
can also improve the appearance of pathologically altered
skin. While the modes of action of most retinoids are not
yet fully understood, it is thought that interactions with
retinoid receptors present throughout the body play a role,
and that side effects occur as a result of the abnormal regulation
of normal metabolic processes. Another example is provided
by calcium- channel blockers, the administration of which
can, for example, cause thickening of the gums (gingivae).
The target cells appear to be fibroblasts in the gums, which
are responsible for the formation of fibrous connnective tissue.
The resulting side effects are primarily attributable to the
upsetting of the balance in collagen formation.
1. Retinoids lead to hypervitaminosis
A syndrome Oral retinoids were first used clinically
at the start of the seventies. Nowadays, this subtance category
is indicated primarily for the treatment of certain skin
disorders. Their effect in acne treatment is based on
a reduction in the secretion and size of the sebaceous glands,
with a corresponding reduction in microbial colonisation and
resulting inflammation. The formation of comedones (black
accumulations of sebum in the secretory ducts of a sebaceous
gland) is also decreased.
Since retinoids are lipophilic, they are stored in the body's
tissues in varying degrees, depending on the dosage and administration
period in each case. Some of the adverse effects are attributable
to their prolonged half-life of several months after completion
of drug administration. The picture of side effects resembles
that seen in chronic hypervitaminosis A and mainly involves
the skin, mucous membranes and bones. The skin, for example,
shows thickening (hyperplasia) of the epidermis, particularly
apparent in the eyelid in laboratory animals. (Hyperplasia
is an enlargement of tissues and organs caused by the abnormal
multiplication of cells). Mucous membranes, for example the
forestomach and oesophagus in rats, can show similar findings.
In the external acoustic meatus, particularly in dogs,
a distinct proliferation of the earwax-forming ceruminal glands,
with consequent increased cerumen production, is observed.
2. Breakdown or excessive formation of bone tissue also
possible
The effects of retinoids on the bones manifest themselves
as a pathologically excessive formation of bone tissue (hyperostosis),
osteoporosis or as disorders of the growth processes
in the epiphyseal plate. The findings differ in their
intensity in the individual animal species investigated to
date.
In dogs and humans, hyperostosis affects primarily the spine,
resulting in changes in shape and size. In humans, hyperostosis
also involves the wrists. Cases of thickening and calcification
of tendons and ligaments, and periosteal detachment have also
been observed. (The periosteum surrounds the bone as a covering
of connective tissue rich in blood vessels and nerves and
containing bone-forming cells, as well as a densely fibrous
outer layer.)
In rats long-term retinoid administration leads to an increased
predisposition to fractures as a result of reduced
bone diameter and thinning of the hard, outer cortical layer
of the bone (Substantia corticalis). In the shaft of a long
bone this is normally several millimetres thick.
In isolated cases, the administration of retinoids to young
animals and children has resulted in delayed growth as a result
of premature epiphyseal plate closure. The epiphyses
are the terminating sections found at the ends of long bones.
The epiphyseal plate is the disk of cartilage between the
epiphysis and metaphysis (section of long bones with a sponge-like
structure) that remains after the ossification of cartilaginous
bone precursors. The plate acts as the bone's growth zone.
Premature closure can result in dwarfism, while the long-term
persistence of the plate can lead to gigantism.
3. Breakdown of the ordered structure of cartilage
The epiphyseal plate consists of various zones: the reserve
zone consists of bluish-milky (hyaline) cartilage in the
resting stage. Cartilage cells (chondrocytes) are a
characteristic feature of cartilage. These are the
more or less rounded cells located in small groups (chondromas)
in the cartilage ground substance, but without any contact
between each other. The reserve zone is bordered by the zone
of proliferation, also known as columnar cartilage
because the chondrocytes are arranged in columns. In the adjacent
resorption zone, the cartilage cells become enlarged, bladder-like,
to form vesicular cartilage. The cartilage cells then die
off in the neighbouring ossification zone.
In this zone, also known as the erosion zone, chondroclasts,
cells that break down cartilage, produce cartilage cavities.
Blood capillaries and cells grow into these cavities and are
transformed into bone-forming osteoblasts (bone progenitor
cells). These attach themselves to the cartilage residues
to form the bone ground substance.
In animals treated with retinoids this ordered structure
of cartilage is broken down. The epiphyseal plate appears
narrowed, and cartilage proliferation is inhibited, expressed
as a significant reduction in columnar cartilage and vesicular
chondrocytes. The erosion zone shows increases in the numbers
of chondroclasts and osteoblasts and haemorrhages. The observed
disruption of growth processes can be severe enough to produce
interruption of the bone plate and the formation of bone chips.
Complete premature closure of the epiphyseal plate, leading
to growth retardation, has even been observed in some cases.
4. Calcium-channel blockers alter the gums
Calcium-channel blockers are preparations that inhibit the
influx of calcium into the cell by blocking various calcium
channels. As a result, they ease the load on the heart,
dilate arterioles and coronary blood vessels, lowering blood
pressure and improving blood flow through the coronary vessels.
They are used primarily in the treatment of hypertension,
angina pectoris and coronary heart disease. The chemical and
pharmacological structures of the substances in this category
can vary considerably.
Although the individual representatives of this group of
substances are well tolerated on the whole, some do produce
side effects, for example an excessive drop in blood pressure,
slowing of the heart rate, headaches or constipation. Numerous
calcium-channel blockers produce macroscopic changes in the
gums. Gum tissue, also known as gingiva, consists of
stratified squamous epithelium on a base of collagenous connective
tissue densely packed with blood vessels and nerves. The administration
of these drugs can lead to thickening of the gums of varying
intensity. The connective tissue section is enlarged and,
in some cases, loosened. A slight increase in the superficial
squamous epithelium is also apparent. In animal experiments,
the picture ranges from slight thickening to the formation
of nodules. This phenomenon is reversed on discontinuation
of the drug.
The mechanism leading to cellular multiplication in the
gums (gingival hyperplasia) is still not fully understood
and involves a complex process affected by numerous factors.
Nor do all inviduals react to the same extent to the preparations.
Essentially, the target cells of these effects on the gums
appear to be the fibroblasts, cells responsible for
the formation of fibrous connective tissue. Interactions with
the respective calcium-channel blockers or their metabolites
probably disrupt the equilibrium of collagen formation.
Genetic factors and plaque-induced inflammation of the gums
also play important roles.
5. Standardisation is an essential precondition for evaluation
An important precondition for all tissue section investigations
is a standardised procedure for the individual steps
and evaluation of the preparations. The procedure should include,
for example, guidelines on isolating the organ and the type
of steps to be performed, the number of sections and the direction
of cut specified for the organ, the anatomical region from
which a sample should be taken, the size of the organ or tissue
sample and the orientation of the tissue preparation on the
slide.
An important development in toxicological histopathology
is the RITA Project (Registry of Industrial Toxicology
Animal Data) initiated by the Hanover-based Fraunhofer
Institute of Toxicology and Aerosol Research in 1988. The
project concerns a database for the collection of validated
histopathology data on tumours and pre-neoplastic lesions
(new tissue formations that tend to lead to tumours) observed
in laboratory rodents such as mice and rats.
To date over 20 chemical/pharmaceutical companies, including
Roche, and institutes in seven European countries are participating
in this project. In fact, study groups involved in RITA across
Europe have, for many years, been drawing up and specifying
standardised techniques for the preparation of histological
sections for all organs, and have also been standardising
the names of, and diagnostic criteria for, the various clinical
syndromes.
Cross-section through the oral cavity of a rat
Cross-section through oral cavity (molar and premolar level)
A Control
B Treatment with high-dose calcium antagonist, showing
greatly thickened gum (outlined in black), due primarily to
connective tissue proliferation (S).
Sections stained with hematoxylin-eosin
Source: Dr. Barbara Lenz, PRNS, Roche
Detail of the epiphyseal plate
Detail of the epiphyseal plate of a rat femur (E)
A Control
B Retinoid treatment, showing loss of orderly cartilage
structrue and narrowing of the epiphyseal plate.
Sections stained with hematoxylin-eosin
Source: Dr. Barbara Lenz, PRNS, Roche
Examples of organ sampling guidelines
A Brain
B Liver
C Gastrointestinal tract
D Larynx
(thick bars = planes of section)
Source: RITA - (Registry of Industrial Toxicology Animal-data)
- Guides for organ sampling and trimming procedures in rats;
Exp Toxic Pathol 1995; 47 (4), 247-266
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