ENU, also known as N-ethyl-N-nitrosourea (chemical formula C<sub>3</sub>H<sub>7</sub>N<sub>3</sub>O<sub>2</sub>), is a highly potent mutagen. For a given gene in mice, ENU can induce 1 new mutation in every 700 loci. It is also toxic at high doses.
The chemical is an alkylating agent, and acts by transferring the ethyl group of ENU to nucleobases (usually thymine) in nucleic acids. Its main targets are the spermatogonial stem cells, from which mature sperm are derived.
Background of discovery of ENU as a mutagen
Bill Russell (1951) created a landmark in the field of mouse genetics by creating a specifically designed mouse strain, the T (test) stock that was used in genetic screens for testing mutagens such as radiations and chemicals. The T-stock mouse harbors 7 recessive, viable mutations affecting easily recognizable traits. At the Oak Ridge National Laboratory, Russell's initial goal was to determine the rate of inheritable gene mutations in the germ line induced by radiations. Thus he decided to use T-stock mice in order to define how often a set of loci could be mutated with radiations. Since the mutations in the T-stock mouse were recessive, the progeny would have a wild type phenotype (as a result of crossing a mutant [e.g.s/s mutant male] to a wild type female [+/+]). Thus with any progeny carrying a mutation induced by radiation at one of the 7 loci, would exhibit the mutant phenotype in the first generation itself. This approach, the specific locus test (SLT) allowed Russell to study a wide range of specific mutations and to calculate the mutation rates induced by radiations.
In addition to studying the effect of radiation for SLT, Russell et al. were also interested in studying the effect of chemical mutagens such as procarbazine and ethylnitrosourea for SLT. At that time, procarbazine was the most potent chemical mutagen known to cause a significant spermatogonial mutagenesis in an SLT, although at a rate one-third of that of X-rays. Russell's earlier mutagenesis work on Drosophila using diethylnitrosoamine (DEN) triggered them to use DEN for the SLT. However, DEN needs to be enzymatically converted into an alkylating agent in order to be mutagenic and probably this enzymatic activation was not sufficient in mammals. This could be illustrated by the extremely low mutation rate in mice given by DEN (3 in 60,179 offspring). To overcome this problem, a new mutagen, N-ethyl N-nitrosourea (ENU), an alkylating agent, which does not need to be metabolised, was suggested to be used by Ekkehart Vegel to Russell et al. The ENU (250 mg/kg) induced mice underwent a period of sterility for 10 weeks. After recovery, 90 males were crossed to the T-stock females and 7584 pups were obtained.
To overcome the problem of initial period of sterility, the Russell group showed that instead of injecting one large dose of ENU, a fractionated dose (100 mg/kg) on a weekly schedule permitted a total higher dose (300–400 mg/kg) However it is also shown to cause GC->AT transitions.
- It is known to induce point mutations, which implies that by mapping for the desired phenotype, the researcher can identify a single candidate gene responsible for the phenotype.
- The point mutations are at approximately 1-2 Mb (Mega-base-pair) interval and occur at an approximate rate of 1 per 700 gametes. Delta 1 is a ligand for the Notch receptor. A homozygous loss-of-function of Delta 1 (Dll1<sup>lacZ/lacZ</sup>) is embryonically lethal. ENU-treated mice were crossed to Dll1<sup>lacZ</sup> heterozygotes. 35 mutant lines were generated in G<sub>1</sub> of which 7 revealed modifiers of the Notch signaling pathway.
Sensitized screens
In the case of genetic diseases involving multiple genes, mutations in multiple genes contributes to the progression of a disease. Mutation in just one of these genes however, might not contribute significantly to any phenotype. Such "predisposing genes" can be identified using sensitized screens. In this type of a screen, the genetic or environmental background is modified so as to sensitize the mouse to these changes. The idea is that the predisposing genes can be unraveled on a modified genetic or environmental background.
Rinchik et al. performed a sensitized screen of mouse mutants predisposed to Diabetic nephropathy. Mice were treated with ENU on a sensitized background of type-1 diabetes. These diabetic mice had a dominant Akita mutation in the insulin-2 gene (Ins2<sup>Akita</sup>). These mice developed albuminuria, a phenotype that was not observed in the non-diabetic offsprings.
Stability
Generally speaking, ENU is fairly unstable, which makes it easier to inactivate when used as an experimental mutagen, compared to moderately more stable mutagens like EMS. Pure crystalline ENU is sensitive to light and moisture, so should be stored at in cold and dry conditions, and freshly prepared into solutions when needed.