Detailed Analysis of the HPRT Assay in Mammalian Cell Systems

This report provides a comprehensive overview of the HPRT assay, a critical method in genetic toxicology utilizing mammalian cell systems. The assay's primary function is to detect gene mutations, particularly those induced by chemicals, by measuring forward mutations in reporter genes such as the HPRT gene. The report details the advantages of using mammalian cells over microbial tests, including the similarity of genomic structure and metabolic processes. It explains the HPRT assay's methodology, which involves exposing cells to a test compound and a toxic nucleotide analogue (6-thioguanine) to identify mutations. The report also discusses the HPRT gene's role in the purine salvage pathway, its location on the X chromosome, and its susceptibility to disorders. The HPRT assay is used to detect DNA damage causing gene mutations, providing valuable insights into genetic occurrences and the impact of mutagenic agents. The report includes details on the assay's procedure, the significance of the HPRT enzyme, and its application in understanding genetic mutations.

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Pros of mammalian cell system
Mutations are widely detected employing mammalian cell test systems that may be
utilized with huge efficiency. Cultured mammalian cells in mutagenicity have some merits as
compared to microbial tests and utilize the same techniques. One advantage of using a
mammalian cell system is that the genomic structure (DNA) is absent in bacteria as it is the cells
division apparatus, and mammalian specific cells metabolism may not be replicated in bacteria.
The other advantage of the mammalian cell system is that it allows detecting gene mutations
induced by chemicals. The cell lines utilized in the tests of mutations allow to measure forward
mutations in reporter genes, particularly in the endogenous hypoxanthine-guanine
phosphoribosyl transferase gene (HPRT) in rodent cells, HPRT in the cells of human beings;
jointly called HPRT gene and HPRT test) and the xanthine-guanine phosphoribosyl transferase
transgene. Thus, the HPRT and XPRT mutation tests permit the detection of diverse spectra of
genetic occurrences. Presently, XPRT is less utilized in test as compared to HPRT test for
regulatory reasons (Berg, Braun, Krug & Schrenk, 2015, pp. 47).
HPRT as big gene
The HPRT is a big gene that has been found to be susceptible to getting hits while it has
been established to the best while performing test in male cells since it is X chromosome. One
mutated allele is sufficient to express the mutated form and create a colony. Characteristically
the HPRT assay is performed where the cells are directly exposed to a toxin, and the toxin is
assessed for being genotoxic, which has the capability to mutate the HPRT gene. This implies

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that since HPRT gene is located in the X chromosome it is susceptible to disorders, such as the
Lesch–Nyhan syndrome (LNS) (Fu et al., 2014).
HPRT assay
The HPRT assay measures the capability of a test chemical to cause mutations at the
HPRT locus in the mammalian cells. HPRT is a human enzyme involved in the purine salvage
pathway that recycles guanine to guanosine monophosphate in DNA degradation. It is encoded
by the human HPRT1 gene. The deficiency of HPRT in human beings has been associated with
Lesch-Nyhan disorder, as well as gout because of the greater degree of uric acid in the human
blood (Guigas, Walz, Gräf, Heller, & Greiner, 2017, pp. 5). The HPRT gene codes for the HPRT
enzyme where the gene is situated on the X chromosome and is hemizygous in CHO cells. The
enzyme is entailed in the purine recycling pathway and catalyses the reaction of hypoxanthine or
guanine with 5-phospho-a-D-ribosyl-1-pyrophosphate (PRPP) to generate either guanosine 5’-
monophosphate or inonsine 5’ monophosphate. Therefore, the HPRT gene is located on the X
chromosome of cells of mammals and is utilized a model of a gene to examine gene mutations in
cell lines of mammals. The HPRT assay can detect many chemicals that are capable of causing
DNA damage, which results in gene mutation (Shah et al., 2016, pp. 11).
Why the HPRT assay used and its purpose?
The HPRT assay utilizes cultured human somatic cells to detect mutagenic agents. The
usual purpose of HPRT in cells is to recycle nucleotide bases from degraded DNA. To detect
mutations in the HPRT gene, there is the need for cells to be originally exposed to the test
compound, which is then exposed to a toxic nucleotide analogue, 6-thioguanine (6TG).

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Consequently, when HPRT is non-mutated and working, 6-thioguanine is integrated into the
DNA, which will trigger cell death. Nevertheless, when HPRT is activated by mutations, no 6-
thioguanine is integrated plus the cells live. Thus, the number of existing cells following a
specified period of cell growth after 6-TG exposure mirrors the mutagenicity of the test agent.
Therefore, HPRT has the role of detecting DNA mutations by performing the different tests in
the chosen genes using mammalian cell system (Walmsley & Tate, 2011, S36).
How is HPRT assay conducted?
The HPRT assess methodology is such that mutations that destroy the functionality of the
HPRT gene and protein are detected by positive selection utilizing a toxic analogues, as well as
HPRT- mutants are seen as visible colonies. To measure the mutagenic effect of a substance
using the HPRT assay, the colony creation ability of V79 cells is investigated by adding 6-TGto
the culture medium. The mutagenic substances will cause forward mutation in the gene coding
for the HPRT enzyme. Therefore, the mutations in the gene result in an incorrect expression of
enzyme sequence, which causes its loss of function that is essential for the nucleotide synthesis
via the salvage pathway (Lohr, Raquet & Schrenk, 2010, pp. 559). The cells may either obtain
nucleotides via de-novo biosynthesis or through energy-saving recycling (Salvage Pathway)
utilizing free bases (for example, guanine or adenine). The V79 cells with an intact HPRT gene
integrate the toxic purine base analogue TG into their DNA structure through the Salvage
Pathway leading to inhibition of cellular metabolism, as well as cytotoxicity. The loss of HPRT
enzyme role because of the mutations results in increased de-novo synthesis since the Salvage
Pathway is barred. The affected V79 cells are hence not able to integrate toxic TG into the DNA.
Therefore, mutant cells cannot be able to penetrate in the exist4ence of the TG, where the normal

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cells that have HPRT are not incorporated into the cells (Havla, Hill, Abdel-Rahman & Richter,
2009, pp. 238).

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Bibliography
Berg, K., Braun, C., Krug, I., & Schrenk, D. (2015). ‘Evaluation of the cytotoxic and mutagenic
potential of three ginkgolic acids’. Toxicology, 327(12), 47–52.
Fu, R., Ceballos-Picot, I., Torres, R. J., Larovere, L. E., Yamada, Y., Nguyen, K. V., … Lesch-
Nyhan Disease International Study Group (2014). Genotype-phenotype correlations in
neurogenetics: Lesch-Nyhan disease as a model disorder. Brain : a journal of neurology, 137(Pt
5), 1282–1303.
Guigas, C., Walz, E., Gräf, V., Heller, K., & Greiner, R. (2017). ‘Mutagenicity of silver
nanoparticles in CHO cells dependent on particle surface functionalization and metabolic
activation’. Journal of Nanoparticle Research, 19(6), 1–14.
Havla, J. B., Hill, C. E., Abdel-Rahman, S. Z., & Richter, E. (2009). ‘Evaluation of the
mutagenic effects of myosmine in human lymphocytes using the HPRT gene mutation assay’.
Food & Chemical Toxicology, 47(1), 237–241.
Lohr, C., Raquet, N., & Schrenk, D. (2010). ‘Application of the concept of relative
photomutagenic potencies to selected furocoumarins in V79 cells’. Toxicology in Vitro, 24(2),
558–566.
Shah, U.-K., Seager, A. L., Fowler, P., Doak, S. H., Johnson, G. E., Scott, S. J., … Jenkins, G. J.
S. (2016). ‘A comparison of the genotoxicity of benzo[a]pyrene in four cell lines with differing
metabolic capacity’. Mutation Research/Genetic Toxicology & Environmental Mutagenesis,
808(2), 8–19.

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Walmsley, R., & Tate, M. (2011). ‘An in vitro pig-A mutation assay? Could it be any better or
easier than MLA or HPRT mutation assays?’ Toxicology Letters, 205(8), S36.