University of California Los Angeles Drugs and Its Side Effects Research Paper The following questions are based on topics we covered in class. I encourage

University of California Los Angeles Drugs and Its Side Effects Research Paper The following questions are based on topics we covered in class. I encourage you to search internet and refer to additional literature to come up with possible answers. You may include scheme(s) and/or figure(s) to illustrate a particular point. Please follow the recommended word limits (including the figure captions). Also, note that your answers will be checked for plagiarism. If found guilty, one will receive zero in this exam, and will be reported to the authorities. Question 1. San Diego–based Arena Pharmaceuticals’ anti-obesity drug – Lorcaserin (brand name – Belviq) was pulled from market in Jan 2020 due to concerns over cancer risks in patients. Explain the mode of action of Lorcaserin and its side-effects. This is one of the many anorexic drugs that have been pulled from market due to their adverse-effects (we looked at another example in the class called fen-phen). Summarize in a few sentences how getting a weight-loss treatment approved by the FDA is a little different compared to other drugs. (400 words max) Question 2. Isoprenaline (brand name – Isuprel) is a medication used for the treatment of bradycardia and heart block. Explain the mode of action of Isoprenaline and speculate why it might be more potent that the natural substrate that it mimics. (300 words max) Question 3. In the class, we studied Ames test to test the potential carcinogenicity of a compound. However, not all compounds that give positive Ames test are carcinogenic. For instance, cruciferous vegetables (e.g., broccoli, cabbage, mustard, etc.) will give a false-positive Ames test. Write a short summary of Ames test and discuss some of the mechanisms causing a false positive test, page 53 of the attached paper (400 words max) Ames Te st 51
bacterial cells to low doses of oxidants allow these
cells to adapt to subsequent challenges by higher
doses of oxidants. These pioneering studies have provided the insights and foundation for understanding
how higher organisms such as mammals adapt to oxidant exposure.
Ames (with Lois Swirsky Gold) has been the leader
in painting a broad picture of the wide variety of mutagens and carcinogens to which humans are exposed.
Their carcinogenic potency data base is the definitive
reference source for all animal cancer tests. Their analyses are having an unusual impact on the prevailing
paradigm in the field. They have characterized the
large background of natural mutagens and carcinogens
and thus have put into perspective, in humans, low
exposures to synthetic chemicals, both qualitatively
and in terms of quantitative carcinogenic potency.
Ames and Gold have shown that half of the chemicals
tested in high-dose animal cancer bioassays, whether
synthetic or natural, are classified as carcinogens.
They have critically addressed the reasons for this
high positivity rate and have supported the interpretation that it is a high-dose effect: induced cell division
and cell replacement converting DNA lesions to
mutations. Thus they have made a rigorous and persuasive case that the current practice of linear extrapolation from high-dose animal cancer tests to predict
human risk for low doses of synthetic chemicals has
distorted the perception of hazard and allocation of
resources, a matter of great societal import. Ames has
also provided an intellectual bridge that connects cancer mechanisms to epidemiological results on the role
of diet in the causation and prevention of cancer.
Ames’s recent research showing that deficiencies of
micronutrients such as folic acid are a major cause of
DNA damage in humans, is likely to have a major
impact on health and prevention of cancer. He has
shown that acetyl carnitine and lipoic acid, fed to rats
at high levels, reverse some of the age-related decay of
mitochondria. These compounds may be conditional
micronutrients and thus have a major impact on delaying aging.
Bruce Ames is a Professor of Biochemistry and
Molecular Biology, and Director of the National Institute of Environmental Health Sciences Center, University of California, Berkeley. He is also a Senior
Research Scientist at the Children’s Hospital Oakland
Research Institute. He is a member of the National
Academy of Sciences and was on their Commission on
Life Sciences. He was a member of the board of directors of the National Cancer Institute, the National
Cancer Advisory Board, from 1976 to 1982. His
awards include the General Motors Cancer Research
Foundation Prize (1983), the Tyler Prize for environmental achievement (1985), the Gold Medal Award
of the American Institute of Chemists (1991), the
Glenn Foundation Award of the Gerontological
Society of America (1992), the Honda Prize of the
Honda Foundation, Japan (1996), the Japan Prize
(1997), the Medal of the City of Paris (1998), and the
US National Medal of Science (1998). His 400 publications have resulted in him being consistently
among the few hundred most-cited scientists (in all
fields): 23rd for 1973±84.
See also: Ames Test
Ames Test
J G Hengstler and F Oesch
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1543
The Ames test (Salmonella typhimurium reverse
mutation assay) is a bacterial short-term test for identification of carcinogens using mutagenicity in bacteria
as an endpoint. It includes mammalian metabolism to
activate promutagens. A high but not complete correlation has been found between carcinogenicity in animals and mutagenicity in the Ames test. The latter
detects mutations in a gene of a histidine-requiring
bacterial strain that produces a histidine-independent
strain. The Ames test is one of the most frequently
applied tests in toxicology. Almost all new pharmaceutical substances and chemicals used in industry are
tested by this assay. The Ames test is named after
Bruce N. Ames, University of California, Berkeley,
who developed this mutagenicity test.
Principle and Tester Strains
Several histidine-requiring bacterial strains of Salmonella typhimurium are used for mutagenicity testing.
Each tester strain contains a different type of mutation
in the histidine operon (Table 1). Because of this
mutation, the tester strain is not able to form colonies
on agar without or with only very low histidine
content. If a mutation is induced in this histidinerequiring strain that generates a histidine-independent
strain, for instance by restoration of the wild-type
gene (Figure 1), it will gain the ability to form colonies also on minimal agar. Since a mutation restores
the histidine-independent wild-type phenotype, the
Ames test is classified as a “reverse” mutation assay.
Approximately 109 bacteria are incubated with a
single concentration of a test substance. Although
the probability for reversion to the wild-type is extremely low for a single bacterium, the extremely high
52
Ames Te st
Table 1
Strain
TA
TA
TA
TA
TA
Genotypes of commonly used Salmonella typhimurium tester strainsa
Mutation
98
100
102
1535
1537
Lipopolysaccharide
barrier
Frameshift in hisD3052
Base substitution in hisG46
Base substitution in hisG428
Base substitution in hisG46
Frameshift in hisC3076
rfa
rfa
rfa
rfa
rfa
DNA repair
Resistance
uvrB
pKM101
Ampicillin
Tetracycline
‡
‡
‡
‡
R
R
R
S
S
S
S
R
S
S
a
All strains were originally derived from Salmonella typhimurium LT2. hisD3052, mutation in the hisD gene coding for
histidinol dehydrogenase; hisG46, mutation in the hisG gene coding for the first step in histidine biosynthesis; hisG428, TA
102 contains A-T base pairs at the site of the mutation in hisG in contrast to TA 100 and TA 1535 that contain G-C base
pairs at the site of mutation; rfa, mutation that causes a strong reduction in the lipopolysaccharide layer; uvrB, a gene
involved in DNA excision repair; pKM101, plasmid that increases chemically induced and spontaneous mutagenesis by
enhancement of error-prone DNA repair; R, resistant; S, sensitive.
Chemical mutagens
Salmonella
typhimurium
TA 1535
Wild-type
bacterium
hisG46
Endogenous
processes
hisG46
5′-C T C-3′
3′-G A G-5′
(Leucine)
5′-C C C-3′
3′-G G G-5′
(Proline)
Formation of colonies
on minimal agar
No colony formation on
minimal agar
Figure 1 Genetic basis of the Ames test shown for
test strain Salmonella typhimurium TA 1535. TA 1535
carries an A to G point mutation compared with the
wild-type bacterium. This point mutation causes an
amino acid exchange (leucine versus proline) in the
histidine operon (hisG46). As a consequence, TA 1535 is
not able to perform histidine biosynthesis. A G to A
point mutation restores the wild-type gene and
produces a bacterium that is able to form a colony also
on minimal agar, containing only very small concentrations of histidine.
number of exposed bacteria results in a high probability that a mutagen will cause a reverse mutation to the
histidine prototroph.
Some mutagens induce exclusively specific types of
mutations that can be classified as base exchange and
frameshift mutations. The set of tester strains shown
in Table 1 includes different mutations in the histidine
operon that combined are able to detect most (probably >85%) of all genotoxic carcinogens. In order to
increase their ability to detect mutagens, the Ames
tester strains also contain other mutations. One mutation (rfa) causes partial loss of the lipopolysaccharide
barrier. As a consequence, the permeability of the cell
wall to large molecules is increased. Another advantage
of rfa is that this mutation leads to completely nonpathogenic bacteria. UvrB indicates a deletion of a
gene required for DNA excision repair, resulting in increased sensitivity in detection of many carcinogens.
The deletion excising the uvrB gene extends also
through the gene required for biotin synthesis. Thus,
they also require biotin for growth. Some tester strains
(TA98, TA100, TA102) contain the plasmid pKM101,
which confers ampicillin resistance and increases
the sensitivity to mutagens by enhancement of errorprone DNA repair. Bacteria lack most of the enzymes
required for the activation of promutagens to mammalian carcinogens. A metabolically active fraction of
mammalian liver homogenate is therefore added in the
Ames test.
Specific Techniques
Two versions of the Ames test are usually applied
(Figure 2): the plate incorporation assay, where bacteria and test substance are mixed and immediately
given onto the agar, and the preincubation assay, where
bacteria and test substance are incubated for 1 h at
37 8C before plating them on agar. The preincubation
version is more sensitive for some compounds, but
also more laborious. In toxicological routine, a negative
result in the plate incorporation assay has to be confirmed in a second series; the first may be a plate incorporate, the second a preincubation assay. Recently, a
Ames Te st 53
(A)
Preincubation assay
S9 Mix
Plate incorporation assay
Bacteria
Test
substance
S9 Mix
Bacteria
Top
agar
Test
substance
Incubation 1h,
shaking water bath
Addition of top agar;
plating on
petri dishes
Plating on petri dishes
after addition
of top agar
Incubation
48−72 h
Growth of
colonies
Negative control
Bacteria incubated
with mutagen
Figure 2 (A) Ames test procedure. All incubations
are performed with and without addition of rat liver S9
mix (see Figure 3). (B) A typical result of an Ames test
with tester strain TA 98 without S9 mix. Only a small
number of revertants can be observed in the solvent
control (left side). Colonies on control dishes are a
consequence of spontaneuous mutations due to endogenous processes, such as generation of reactive
oxygen species and physical instability of DNA. Addition
of a mutagen, such as 10 mg benzo [a]pyrene-4,5-oxide
per plate dramatically increases the number of revertants (right side). (Preparation of plates: Hildegard
Georgi; photo: Friedrich Feyrer.)
new version of the Ames test, `Ames II,’ has been
developed as a microwell fluctuation test in contrast
to the standard plate or preincubation test. Ames II
allows automated, high-throughput screening, requiring only very small amounts of test substance.
Metabolizing System: Rat Liver S9 Mix
Since most carcinogens are not carcinogenic directly,
but are active only after metabolism, the compounds
are tested in the Ames test in the presence of a mammalian metabolizing system as well as directly. The
9000 supernatant fraction (`S9′; see Figure 3) of liver
homogenate from rats treated with substances causing
a strong induction of many xenobiotic metabolizing
enzymes (e.g., Aroclor 1254 or a combination of
b-naphthoflavone and phenobarbital) in combination
with an NADPH-generating system have been
shown to be very favorable for activation. NADPH
is required because it represents a cofactor for cytochrome P450-dependent monooxygenase activity.
Liver S9 is highly active in carcinogen metabolism,
since the liver represents the most important organ
for the metabolism of most foreign compounds.
Guidelines for Interpretation of Ames
Test Data
There are several criteria for determining a positive
result, including a reproducible increase in the number
of revertants or a dose-related increase in mutations. A
positive result in the Ames test will usually initiate
additional investigations by other mutagenicity assays
including also mammalian cells. If the positive result is
confirmed, most pharmaceutical companies will terminate further development of a drug. However, an
Ames-positive substance is not necessarily harmful to
humans. Although the Ames test is a useful tool in
screening for potential carcinogens, often false-positive
results are obtained. It is generally accepted that a
substance may be used clinically even if the Ames
test is positive, when the positive effect is due to a
mechanism not relevant for humans and, ideally, if
additional mutagenicity tests with mammalian cells
in vivo and in vitro, tests for chromosomal aberrations, and animal carcinogenicity studies with two
species were negative.
Mechanisms causing false-positive results may be:
(1) differences between bacteria and mammalian cells
concerning metabolism and DNA repair; (2) differences between rat and human liver, since rat liver S9
mix is used in the standard Ames test; and (3) differences between liver homogenate preparations such as
S9 mix and intact hepatocytes. The latter is due to the
loss of barrier effects in homogenate by destruction of
54
Amino A cids
5−7 days
Liver
S9
Administration of substance
that induce liver enzymes
– Excision of liver
– Homogenization
– Centrifugation at 9000g
Sediment
Figure 3 Preparation of rat liver S9 mix. After centrifugation of liver homogenate at 9000, the supernatant (S9) is
used as a metabolizing system in the Ames test. S9 contains microsomes and cytosol and therefore all microsomal and
cytosolic xenobiotic metabolizing enzymes. In contrast, the sediment containing cell membranes and lysosomes is
discarded. An NADPH (cofactor for cytochrome P450-dependent monooxygenase activity)-generating system is
added to S9 to form the “S9 mix.”
the cell membrane and loss of phase II metabolizing
enzymes owing to dilution of cofactors. Thus, bear in
mind that the Ames test is an artificial system and does
not necessarily reflect the in vivo situation. This is
illustrated by the observation that the endogenous
tripeptide glutathione and the amino acid cysteine
both are positive in the Ames test under specific conditions (Glatt et al., 1983).
Performance and interpretation of Ames test results
have been standardized by international guidelines,
such as those of the Organization for Economic Cooperation and Development (OECD Guideline 471)
and the International Conference on Harmonization
(ICH).
Future Prospects
The Ames test is a sensitive tool in screening for
potential genotoxic carcinogens. However, despite the
high correlation, a positive result is difficult to interpret
for the individual case in question, because a mutagen
in the Ames test is not necessarily harmful to humans.
These problems can be alleviated in future. It has been
shown clearly that the use of intact cells instead of S9
mix improves the correlation between carcinogenicity
and mutagenicity data (Utesch et al., 1987). Since
cryopreserved human hepatocytes are now available,
they can be used as a metabolizing system instead of
rat S9 mix (Hengstler et al., 2000). This gives a possibility to test whether a positive result in the standard
rat S9 Ames test is also relevant to humans.
Further Reading
Ames B and Hooper K (1978) Does carcinogenic potency
correlate with mutagenic potency in the Ames assay? Nature
274: 19±20.
Ames BN (1979) Identifying environmental chemicals causing
mutations and cancer. Science 204: 587±593.
References
Glatt H, Protic-Sabljic M and Oesch F (1983) Mutagenicity of
glutathione and cysteine in the Ames test. Science 220:
961±963.
Hengstler JG, Utesch D, Steinberg P et al. (2000) Cryopreserved primary hepatocytes as a constantly available in
vitro model for the evaluation of human and animal drug
metabolism and enzyme induction. Drug Metabolism Review
32: 81±118.
Utesch D, Glatt H and Oesch F (1987) Rat hepatocytemediated bacterial mutagenicity in relation to the carcinogenic potency of benz(a)anthracene, benzo(a)pyrene, and
twenty-five methylated derivatives. Cancer Research 47:
1509±1515.
See also: Ames, Bruce; Mutagens; Reverse
Mutation
Amino Acids
L B Willis, P A Lessard and A J Sinskey
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.0041
Amino acids are a class of important biomolecules that
contain both amino groups (-NH3‡) and carboxylate
groups (-COO ). In most contexts, the term `amino
acids’ refers to the a-amino acids, so-called because
both the amino and carboxyl groups are attached to
the a-carbon of the structure depicted in Figure 1A.
However, other types of amino acids are encountered

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